Angiotensin in treating brain conditions

ABSTRACT

The present invention provides, among other things, methods and compositions for treating brain conditions. In some embodiments, the methods include administering to a subject suffering from or susceptible to a brain condition an angiotensin (1-7) peptide via either an intravenous or subcutaneous route of administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/752,202, filed Jan. 28, 2013, which claims priority fromU.S. provisional patent application Ser. No. 61/708,793, filed Oct. 2,2012, and U.S. provisional patent application Ser. No. 61/720,299, filedOct. 30, 2012, the disclosures of which are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listingsubmitted electronically as a .txt file named “2009912-0053_ST25” onJan. 28, 2012. The .txt file was generated on Jan. 28, 2013 and is 11 kbin size. The entire contents of the Sequence Listing are hereinincorporated by reference.

BACKGROUND

Proper functioning of the central nervous system is essential in anyanimal. Damage to the brain in particular, such as through an ischemicor hemorrhagic stroke, can have dramatic and potentially deadly effects.One obstacle to treatment or prevention of brain damaging events is theblood brain barrier, which is a collection of tight junctions betweenneighboring capillary endothelial cells of the brain. These junctionsprevent most substances from crossing unless they are either highlylipophilic or specifically transported across the blood brain barrier.As a result, it is extremely difficult to administer therapeutics viatraditionally preferred routes, such as through an intravenous orsubcutaneous administration and observe a therapeutic effect in thebrain.

SUMMARY OF THE INVENTION

The present invention provides, among other things, methods andcompositions for treating brain conditions including, but not limitedto: stroke, vascular dementia, and traumatic brain injury. As describedin the Examples section below, the present invention is, in part, basedon the surprising discovery that systemic administration, such assubcutaneous administration, of an angiotensin (1-7) peptide (e.g.,PanCyte), results in improvement of neurological and motor function in arat model of ischemic stroke. Prior to the present invention, it wasbelieved that angiotensin(1-7) would not cross the blood brain barrierand thus had to be administered intracerebrovascularly (ICV) or usingcomplex methods such as infection of hematopoietic stem cells, which arecapable of crossing the blood brain barrier, with a lentivirus thatcauses overexpression of Ang(1-7). Mecca et al., Cerebroprotection byAngiotensin-(1-7) in Endothelin-1-Induced Ischaemic Stroke, (2011) ExpPhysiol. 2011 96(10):1084-1096. No one had shown that administration ofan angiotensin (1-7) peptide or a non-peptidic Angiotensin-(1-7) agonistvia a systemic (for example, either a subcutaneous or intravenous) routecould result in therapeutic levels reaching the brain and, inparticular, damaged brain tissue.

In some embodiments, the invention provides methods of treating a braincondition including administering to a subject suffering from orsusceptible to a brain condition an angiotensin (1-7) peptide viasystemic administration. In some embodiments, systemic administrationsuitable for the present invention is intravenous administration. Insome embodiments, systemic administration suitable for the presentinvention is subcutaneous administration. In some embodiments, systemicadministration suitable for the present invention is oraladministration. In some embodiments, systemic administration suitablefor the present invention does not include intracerebroventricularadministration. In some embodiments, the brain condition is selectedfrom stroke, vascular dementia, and traumatic brain injury. In someembodiments, the stroke is ischemic stroke. In some embodiments, thestroke is hemorrhagic stroke.

In some embodiments, the angiotensin (1-7) peptide is administered viacontinuous infusion. In some embodiments, the angiotensin (1-7) peptideis administered at an administration interval. For example, theangiotensin (1-7) peptide may be administered three times a day, twice aday, once per day, twice per week, once per week, three times per month,twice per month, once every two weeks, once every three weeks, onceevery four weeks, once a month, once every two months, once every threemonths, once every four months, once every five months, once every sixmonths, at an irregular interval.

It is contemplated that various embodiments may use different amounts ofangiotensin (1-7) peptide. In some embodiments, the angiotensin (1-7)peptide is administered at an effective dose ranging from about 1-1,000μg/kg/day (e.g., ranging from about 1-900 μg/kg/day, 1-800 μg/kg/day,1-700 μg/kg/day, 1-600 μg/kg/day, 1-500 μg/kg/day, 1-400 μg/kg/day,1-300 μg/kg/day, 1-200 μg/kg/day, 1-100 μg/kg/day, 1-90 μg/kg/day, 1-80μg/kg/day, 1-70 μg/kg/day, 1-60 μg/kg/day, 1-50 μg/kg/day, 1-40μg/kg/day, 1-30 μg/kg/day, 1-20 μg/kg/day, 1-10 μg/kg/day). In someembodiments, the angiotensin (1-7) peptide is administered at aneffective dose ranging from about 1-500 μg/kg/day. In some embodiments,the angiotensin (1-7) peptide is administered at an effective doseranging from about 1-100 μg/kg/day. In some embodiments, the angiotensin(1-7) peptide is administered at an effective dose ranging from about1-60 μg/kg/day. In some embodiments, the angiotensin (1-7) peptide isadministered at an effective dose selected from about 1, 2, 4, 6, 8, 10,15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000ug/kg/day.

It is also contemplated that various angiotensin (1-7) peptides may beused in various embodiments. In some embodiments, the angiotensin (1-7)peptide comprises the naturally-occurring Angiotensin (1-7) amino acidsequence of Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO:1).

In some embodiments, the angiotensin (1-7) peptide is a functionalequivalent of SEQ ID NO:1. In some embodiments, the functionalequivalent is a linear peptide. In some embodiments, the linear peptidecomprises a sequence that includes at least four, at least five, or atleast six amino acids from the seven amino acids that appear in thenaturally-occurring Angiotensin (1-7), wherein the at least four, fiveor six amino acids maintain their relative positions as they appear inthe naturally-occurring Angiotensin (1-7). In some embodiments, thelinear peptide contains 4-25 amino acids. In some embodiments, thelinear peptide is a fragment of the naturally-occurring Angiotensin(1-7). In some embodiments, the linear peptide contains amino acidsubstitutions, deletions and/or insertions in the naturally-occurringAngiotensin (1-7). In some embodiments, the linear peptide has an aminoacid sequence of Asp¹-Arg²-Val³-ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO:2).

In some embodiments, the functional equivalent is a cyclic peptide. Insome embodiments, the cyclic peptide comprises a linkage between aminoacids. In some embodiments, the linkage is located at residuescorresponding to positions Tyr⁴ and Pro⁷ in naturally-occurringAngiotensin (1-7). In some embodiments, the linkage is a thioetherbridge. In some embodiments, the cyclic peptide comprises an amino acidsequence otherwise identical to the naturally-occurring Angiotensin(1-7) amino acid sequence of Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ IDNO:1). In some embodiments, the cyclic peptide comprises a norleucine(Nle) replacing position Val³ in naturally-occurring Angiotensin (1-7).In some embodiments, the cyclic peptide is a 4,7-cyclized angiotensin(1-7) with the following formula Asp¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQID NO: 22). In some embodiments, the cyclic peptide is a 4,7-cyclizedangiotensin (1-7) with the following formula:

In some embodiments, the angiotensin (1-7) peptide comprises one or morechemical modifications to increase protease resistance, serum stabilityand/or bioavailability. In some embodiments, the one or more chemicalmodifications comprise pegylation.

In some embodiments, the present invention provides methods of treatingbrain conditions including, but not limited to: stroke, vasculardementia, and traumatic brain injury including administering to asubject who is suffering from or susceptible to one or more brainconditions an angiotensin (1-7) receptor agonist. In some embodiments,the angiotensin (1-7) receptor agonist is a non-peptidic agonist. Insome embodiments, the non-peptidic agonist is a compound with thefollowing structure:

or a pharmaceutically acceptable salt thereof.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an exemplary bar graph comparing the results of a step testadministered on rats who received a transient middle cerebral arterialocclusion and either some amount of PanCyte for 14 or 49 days.

FIG. 2 shows an exemplary bar graph comparing the results of a forelimbplacement test administered on rats who received a transient middlecerebral arterial occlusion and either some amount of PanCyte for 14 or49 days.

FIG. 3 shows an exemplary bar graph comparing the results of a bodyswing test administered on rats who received a transient middle cerebralarterial occlusion and either some amount of PanCyte for 14 or 49 days.

FIG. 4 shows an exemplary bar graph comparing the results of aneurological test (modified Neuroscore Scoring Scale) administered onrats who received a transient middle cerebral arterial occlusion andeither some amount of PanCyte for 14 or 49 days.

FIG. 5 shows an exemplary bar graph comparing the results of a step testadministered on rats who received a transient middle cerebral arterialocclusion and either subcutaneously administered TXA127, PanCyte, orlinear PanCyte for 28 days.

FIG. 6 shows an exemplary bar graph comparing the results of a forelimbplacement test administered on rats who received a transient middlecerebral arterial occlusion and either subcutaneously administeredTXA127, PanCyte, or linear PanCyte for 28 days.

FIG. 7 shows an exemplary bar graph comparing the results of a bodyswing test administered on rats who received a transient middle cerebralarterial occlusion and either subcutaneously administered TXA127,PanCyte, or linear PanCyte for 28 days.

FIG. 8 shows an exemplary bar graph comparing the results of aneurological test (modified Neuroscore Scoring Scale) administered onrats who received a transient middle cerebral arterial occlusion andeither subcutaneously administered TXA127, PanCyte, or linear PanCytefor 28 days.

FIG. 9 shows an exemplary bar graph comparing the blood perfusion ratiobetween ipsilateral and contralateral sides, as well as blood vesseldiameter, in animals subcutaneously administered TXA127, PanCyte, orlinear PanCyte via injection for 28 days.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In particularembodiments, where a peptide is biologically active, a portion of thatpeptide that shares at least one biological activity of the peptide istypically referred to as a “biologically active” portion. In certainembodiments, a peptide has no intrinsic biological activity but thatinhibits the effects of one or more naturally-occurring angiotensincompounds is considered to be biologically active.

Brain Condition—as used herein, a “brain condition” is any disease,disorder or event that results in damage and/or dysfunction of at leasta portion of a subject's brain. Non-limiting examples of brainconditions include: stroke (both ischemic and hemorrhagic), vasculardementia, and traumatic brain injury.

Carrier or diluent: As used herein, the terms “carrier” and “diluent”refers to a pharmaceutically acceptable (e.g., safe and non-toxic foradministration to a human) carrier or diluting substance useful for thepreparation of a pharmaceutical formulation. Exemplary diluents includesterile water, bacteriostatic water for injection (BWFI), a pH bufferedsolution (e.g. phosphate-buffered saline), sterile saline solution,Ringer's solution or dextrose solution.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic agent for thepatient to be treated. Each unit contains a predetermined quantity ofactive material calculated to produce the desired therapeutic effect. Itwill be understood, however, that the total dosage of the compositionwill be decided by the attending physician within the scope of soundmedical judgment.

Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as thatterm is used herein, is a set of unit doses (typically more than one)that are administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regime comprises a plurality ofdoses and at least two different time periods separating individualdoses. In some embodiments, the therapeutic agent is administeredcontinuously over a predetermined period. In some embodiments, thetherapeutic agent is administered once a day (QD) or twice a day (BID).

Functional equivalent or derivative: As used herein, the term“functional equivalent” or “functional derivative” denotes, in thecontext of a functional derivative of an amino acid sequence, a moleculethat retains a biological activity (either function or structural) thatis substantially similar to that of the original sequence. A functionalderivative or equivalent may be a natural derivative or is preparedsynthetically. Exemplary functional derivatives include amino acidsequences having substitutions, deletions, or additions of one or moreamino acids, provided that the biological activity of the protein isconserved. The substituting amino acid desirably has chemico-physicalproperties which are similar to that of the substituted amino acid.Desirable similar chemico-physical properties include, similarities incharge, bulkiness, hydrophobicity, hydrophilicity, and the like.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control subject (or multiple control subject) inthe absence of the treatment described herein. A “control subject” is asubject afflicted with the same form of disease as the subject beingtreated, who is about the same age as the subject being treated.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, substantially 100%, or 100% pure. Asused herein, a substance is “pure” if it is substantially free of othercomponents. As used herein, the term “isolated cell” refers to a cellnot contained in a multi-cellular organism.

Prevent: As used herein, the term “prevent” or “prevention”, when usedin connection with the occurrence of a disease, disorder, and/orcondition, refers to reducing the risk of developing the disease,disorder and/or condition. See the definition of “risk.”

Polypeptide: The term “polypeptide” as used herein refers a sequentialchain of amino acids linked together via peptide bonds. The term is usedto refer to an amino acid chain of any length, but one of ordinary skillin the art will understand that the term is not limited to lengthychains and can refer to a minimal chain comprising two amino acidslinked together via a peptide bond. As is known to those skilled in theart, polypeptides may be processed and/or modified.

Protein: The term “protein” as used herein refers to one or morepolypeptides that function as a discrete unit. If a single polypeptideis the discrete functioning unit and does not require permanent ortemporary physical association with other polypeptides in order to formthe discrete functioning unit, the terms “polypeptide” and “protein” maybe used interchangeably. If the discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” refers to the multiple polypeptides that arephysically coupled and function together as the discrete unit.

Risk: As will be understood from context, a “risk” of a disease,disorder, and/or condition comprises a likelihood that a particularindividual will develop a disease, disorder, and/or condition (e.g.,stroke). In some embodiments, risk is expressed as a percentage. In someembodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed asa risk relative to a risk associated with a reference sample or group ofreference samples. In some embodiments, a reference sample or group ofreference samples have a known risk of a disease, disorder, conditionand/or event (e.g., stroke). In some embodiments a reference sample orgroup of reference samples are from individuals comparable to aparticular individual. In some embodiments, relative risk is 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more.

Stability: As used herein, the term “stable” refers to the ability ofthe therapeutic agent to maintain its therapeutic efficacy (e.g., all orthe majority of its intended biological activity and/or physiochemicalintegrity) over extended periods of time. The stability of a therapeuticagent, and the capability of the pharmaceutical composition to maintainstability of such therapeutic agent, may be assessed over extendedperiods of time (e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 monthsor more). In certain embodiments, pharmaceutical compositions describedherein have been formulated such that they are capable of stabilizing,or alternatively slowing or preventing the degradation, of one or moretherapeutic agents formulated therewith. In the context of a formulationa stable formulation is one in which the therapeutic agent thereinessentially retains its physical and/or chemical integrity andbiological activity upon storage and during processes (such asfreeze/thaw, mechanical mixing and lyophilization).

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of the disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition may not exhibitsymptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,condition, or event (for example, ischemic stroke) may be characterizedby one or more of the following: (1) a genetic mutation associated withdevelopment of the disease, disorder, and/or condition; (2) a geneticpolymorphism associated with development of the disease, disorder,and/or condition; (3) increased and/or decreased expression and/oractivity of a protein associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, condition, and/or event (5) having undergone,planning to undergo, or requiring a transplant. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionwill develop the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition will not develop the disease, disorder, and/orcondition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides, among other things, improvedcompositions and methods for treating or reducing the risk of brainconditions resulting from damage to or disorder of brain tissue.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Brain Conditions

Stroke

The brain is highly vulnerable to a disturbance in its oxygen supply.Anoxia and ischemia lasting only a few seconds can cause symptoms and ifthe condition persists for minutes, they can cause irreversible neuronaldamage. Accordingly, stroke is a prominent cause of serious, long-termdisability and a leading cause of death in the United States. Stroke isalso a significant burden on the medical industry, with total healthcosts for disability due to stroke being estimated at approximately $53billion annually.

There are two types of stroke: ischemic and hemorrhagic. Ischemic strokeinvolves an obstruction in one or more blood vessels that supply bloodto brain tissue, for example, occlusion resulting from atheroscleroticthrombii, or embolism. Ischemic stroke (cerebral ischemia) representapproximately 88% of all strokes, making ischemic stroke one of the mostcommon types of cerebrovascular injury. Ischemic conditions in the brainquickly lead to neuronal death, often leading to permanent sensorimotordeficits. A hemorrhagic stroke is defined herein as the accumulation ofblood anywhere within the cranial vault. Hemorrhagic strokes may resultfrom many causes, including injury resulting from an expanding hematoma,which can disrupt or distort tissue.

A major barrier in the treatment of both ischemic and hemorrhagic strokeis delivery of a therapeutic that will reach affected tissue. Given theeffectiveness of the blood brain barrier, few compounds are capable ofcrossing into and affecting cerebral tissue. Previously, delivery ofcompounds such as angiotensin (1-7), had to be made usingintracerebroventricular (ICV) delivery. Surprisingly, embodiments of thepresent invention, including the exemplary angiotensin (1-7) peptidesdescribed below, are able to cross the blood brain barrier withoutcomplex delivery systems such as modified stem cells or the like.Rather, in some embodiments, angiotensin (1-7) peptides may be deliveredvia intravenous or subcutaneous routes.

Vascular Dementia

Vascular dementia is the second most common form of dementia, behindAlzheimer's Disease. Vascular dementia may result from problems withblood supply in the brain, such as those caused by ischemic orhemorrhagic stroke or from other causes that lead to development oflesions in the brain. Other causes of vascular dementia include cerebralamyloid angiopathy, hypercholesterolemia, diabetes mellitus, orcardiovascular disease. Dementia resulting from one or more strokes isalso known as “single-infarct dementia” or “multi-infarct dementia,”depending upon the root cause.

Treatment of vascular dementia has primarily focused on the preventionof further cerebrovascular lesions through use of antiplatelet drugs andlife style changes (alteration of diet, cessation of smoking, etc).Cholinesterase inhibitors such as galantamine have also been exploredfor use in this clinical scenario, but this type of treatment isconcerned with maintenance of acetylcholine function in the brain, andnot recovery or generation of an improved and sustained blood supply.Thus, embodiments of the present invention represent a novel intravenousand subcutaneous therapy targeted to improving the underlying causes ofthe disease rather than management of symptoms or maximization ofremaining tissue resources.

Traumatic Brain Injury

Traumatic brain injury (TBI), a form of acquired brain injury, occurswhen a sudden trauma causes damage to the brain. TBI can result when thehead suddenly and violently hits an object (or vice versa), or when anobject pierces the skull and enters brain tissue. Symptoms of a TBI canbe mild, moderate, or severe, depending on the extent of the damage tothe brain. A person with a mild TBI may remain conscious or mayexperience a loss of consciousness for a few seconds or minutes. Othersymptoms of mild TBI include headache, confusion, lightheadedness,dizziness, blurred vision or tired eyes, ringing in the ears, bad tastein the mouth, fatigue or lethargy, a change in sleep patterns,behavioral or mood changes, and trouble with memory, concentration,attention, or thinking. A person with a moderate or severe TBI may showthese same symptoms, but may also have a headache that gets worse ordoes not go away, repeated vomiting or nausea, convulsions or seizures,an inability to awaken from sleep, dilation of one or both pupils of theeyes, slurred speech, weakness or numbness in the extremities, loss ofcoordination, and increased confusion, restlessness, or agitation.

Treatments for TBI focus primarily on preventing further injury orcomplications. Primary concerns in treating TBI include insuring properoxygen supply to the brain and the rest of the body, maintainingadequate blood flow, and controlling blood pressure. Often, the primarytreatment a TBI sufferer receives post-stabilization is rehabilitationthat involves individually tailored treatment programs in the areas ofphysical therapy, occupational therapy, speech/language therapy,psychology/psychiatry, and social support. Embodiments of the presentinvention provide a novel treatment for these patients.

Angiotensin (1-7) Peptides

As used herein, the term “angiotensin (1-7) peptide” refers to bothnaturally-occurring Angiotensin (1-7) and any functional equivalent,analogue or derivative of naturally-occurring Angiotensin (1-7). As usedherein, “peptide” and “polypeptide” are interchangeable terms and referto two or more amino acids bound together by a peptide bond. As usedherein, the terms “peptide” and “polypeptide” include both linear andcyclic peptide. The terms “angiotensin-(1-7)”, “Angiotensin-(1-7)”, and“Ang-(1-7)” are used interchangeably.

Naturally-Occurring Angiotensin (1-7)

Naturally-occurring Angiotensin (1-7) (also referred to as Ang-(1-7)) isa seven amino acid peptide shown below:

Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO: 1)It is part of the renin-angiotensin system and is converted from aprecursor, also known as Angiotensinogen, which is an α-2-globulin thatis produced constitutively and released into the circulation mainly bythe liver. Angiotensinogen is a member of the serpin family and alsoknown as renin substrate. Human angiotensinogen is 452 amino acids long,but other species have angiotensinogen of varying sizes. Typically, thefirst 12 amino acids are the most important for angiotensin activity:

(SEQ ID NO: 3) Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷-Phe⁸-His⁹-Leu¹⁰-Val¹¹-Ile¹²

Different types of angiotensin may be formed by the action of variousenzymes. For example, Angiotensin (1-7) is generated by action ofAngiotensin-converting enzyme 2 (ACE 2).

Ang-(1-7) is an endogenous ligand for Mas receptors. Mas receptors areG-protein coupled receptor containing seven transmembrane spanningregions. As used herein, the term “angiotensin-(1-7) receptor’encompasses the G Protein-Coupled Mas Receptors.

As used herein, the term “naturally-occurring Angiotensin (1-7)”includes any Angiotensin (1-7) peptide purified from natural sources andany recombinantly produced or chemically synthesized peptides that havean amino acid sequence identical to that of the naturally-occurringAngiotensin (1-7).

Functional Equivalents, Analogs or Derivatives of Ang-(1-7)

In some embodiments, an angiotensin (1-7) peptide suitable for thepresent invention is a functional equivalent of naturally-occurringAng-(1-7). As used herein, a functional equivalent ofnaturally-occurring Ang-(1-7) refers to any peptide that shares aminoacid sequence identity to the naturally-occurring Ang-(1-7) and retainsubstantially the same or similar activity as the naturally-occurringAng-(1-7). For example, in some embodiments, a functional equivalent ofnaturally-occurring Ang-(1-7) described herein has pro-angiogenicactivity as determined using methods described herein or known in theart, or an activity such as nitric oxide release, vasodilation, improvedendothelial function, antidiuresis, or one of the other propertiesdiscussed herein, that positively impacts angiogenesis. In someembodiments, a functional equivalent of naturally-occurring Ang-(1-7)described herein can bind to or activate an angiotensin-(1-7) receptor(e.g., the G protein-coupled Mas receptor) as determined using variousassays described herein or known in the art. In some embodiments, afunctional equivalent of Ang-(1-7) is also referred to as an angiotensin(1-7) analogue or derivative, or functional derivative.

Typically, a functional equivalent of angiotensin (1-7) shares aminoacid sequence similarity to the naturally-occurring Ang-(1-7). In someembodiments, a functional equivalent of Ang-(1-7) according to theinvention contains a sequence that includes at least 3 (e.g., at least4, at least 5, at least 6, at least 7) amino acids from the seven aminoacids that appear in the naturally-occurring Ang-(1-7), wherein the atleast 3 (e.g., at least 4, at least 5, at least 6, or at least 7) aminoacids maintain their relative positions and/or spacing as they appear inthe naturally-occurring Ang-(1-7).

In some embodiments, a functional equivalent of Ang-(1-7) also encompassany peptide that contain a sequence at least 50% (e.g., at least 60%,70%, 80%, or 90%) identical to the amino acid sequence ofnaturally-occurring Ang-(1-7). Percentage of amino acid sequenceidentity can be determined by alignment of amino acid sequences.Alignment of amino acid sequences can be achieved in various ways thatare within the skill in the art, for instance, using publicly availablecomputer software such as BLAST, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared.Preferably, the WU-BLAST-2 software is used to determine amino acidsequence identity (Altschul et al., Methods in Enzymology 266, 460-480(1996); blast.wustl/edu/blast/README.html). WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. HSP score (S) and HSP S2parameters are dynamic values and are established by the program itself,depending upon the composition of the particular sequence, however, theminimum values may be adjusted and are set as indicated above.

In some embodiments, a functional equivalent, analogue or derivative ofAng-(1-7) is a fragment of the naturally-occurring Ang-(1-7). In someembodiments, a functional equivalent, analogue or derivative ofAng-(1-7) contains amino acid substitutions, deletions and/or insertionsin the naturally-occurring Ang-(1-7). Ang-(1-7) functional equivalents,analogues or derivatives can be made by altering the amino acidsequences by substitutions, additions, and/or deletions. For example,one or more amino acid residues within the sequence of thenaturally-occurring Ang-(1-7) (SEQ ID NO:1) can be substituted byanother amino acid of a similar polarity, which acts as a functionalequivalent, resulting in a silent alteration. Substitution for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the positively charged(basic) amino acids include arginine, lysine, and histidine. Thenonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine,phenylalanine, valine, proline, tryptophane, and methionine. Theuncharged polar amino acids include serine, threonine, cysteine,tyrosine, asparagine, and glutamine. The negatively charged (acid) aminoacids include glutamic acid and aspartic acid. The amino acid glycinemay be included in either the nonpolar amino acid family or theuncharged (neutral) polar amino acid family. Substitutions made within afamily of amino acids are generally understood to be conservativesubstitutions. For example, the amino acid sequence of a peptideinhibitor can be modified or substituted.

Examples of Ang-(1-7) functional equivalents, analogues and derivativesare described in the section entitled “Exemplary Angiotensin(1-7)Peptides” below.

An angiotensin-(1-7) peptide can be of any length. In some embodiments,an angiotensin-(1-7) peptide according to the present invention cancontain, for example, from 4-25 amino acids (e.g., 4-20, 4-15, 4-14,4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7 amino acids). In some embodiments,the linear peptide contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

In some embodiments, an angiotensin-(1-7) peptide contains one or moremodifications to increase protease resistance, serum stability and/orbioavailability. In some embodiments, suitable modifications areselected from pegylation, acetylation, glycosylation, biotinylation,substitution with D-amino acid and/or un-natural amino acid, and/orcyclization of the peptide.

As used herein, the term “amino acid,” in its broadest sense, refers toany compound and/or substance that can be incorporated into apolypeptide chain. In certain embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In certain embodiments, an amino acid is anaturally-occurring amino acid. In certain embodiments, an amino acid isa synthetic or un-natural amino acid (e.g., α,α-disubstituted aminoacids, N-alkyl amino acids); in some embodiments, an amino acid is ad-amino acid; in certain embodiments, an amino acid is an 1-amino acid.“Standard amino acid” refers to any of the twenty standard amino acidscommonly found in naturally occurring peptides including both l- andd-amino acids which are both incorporated in peptides in nature.“Nonstandard” or “unconventional amino acid” refers to any amino acid,other than the standard amino acids, regardless of whether it isprepared synthetically or obtained from a natural source. As usedherein, “synthetic or un-natural amino acid” encompasses chemicallymodified amino acids, including but not limited to salts, amino acidderivatives (such as amides), and/or substitutions. Amino acids,including carboxy- and/or amino-terminal amino acids in peptides, can bemodified by methylation, amidation, acetylation, and/or substitutionwith other chemical groups that can change the peptide's circulatinghalf-life without adversely affecting its activity. Examples ofunconventional or un-natural amino acids include, but are not limitedto, citrulline, ornithine, norleucine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methylthreonine (MeBmt), N-methyl-leucine(MeLeu), aminoisobutyric acid, statine, and N-methyl-alanine (MeAla).Amino acids may participate in a disulfide bond. The term “amino acid”is used interchangeably with “amino acid residue,” and may refer to afree amino acid and/or to an amino acid residue of a peptide. It will beapparent from the context in which the term is used whether it refers toa free amino acid or a residue of a peptide.

In certain embodiments, angiotensin-(1-7) peptides contain one or moreL-amino acids, D-amino acids, and/or un-natural amino acids.

In addition to peptides containing only naturally occurring amino acids,peptidomimetics or peptide analogs are also encompassed by the presentinvention. Peptide analogs are commonly used in the pharmaceuticalindustry as non-peptide drugs with properties analogous to those of thetemplate peptide. The non-peptide compounds are termed “peptidemimetics” or peptidomimetics (Fauchere et al., Infect. Immun. 54:283-287(1986); Evans et al., J. Med. Chem. 30:1229-1239 (1987)). Peptidemimetics that are structurally related to therapeutically usefulpeptides and may be used to produce an equivalent or enhancedtherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to the paradigm polypeptide (i.e., a polypeptidethat has a biological or pharmacological activity) such asnaturally-occurring receptor-binding polypeptides, but have one or morepeptide linkages optionally replaced by linkages such as —CH₂NH—,—CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans), —CH₂SO—, —CH(OH)CH₂—,—COCH₂— etc., by methods well known in the art (Spatola, PeptideBackbone Modifications, Vega Data, 1(3):267 (1983); Spatola et al. LifeSci. 38:1243-1249 (1986); Hudson et al. Int. J. Pept. Res. 14:177-185(1979); and Weinstein. B., 1983, Chemistry and Biochemistry, of AminoAcids, Peptides and Proteins, Weinstein eds, Marcel Dekker, New-York,).Such peptide mimetics may have significant advantages overnaturally-occurring polypeptides including more economical production,greater chemical stability, enhanced pharmacological properties (e.g.,half-life, absorption, potency, efficiency, etc.), reduced antigenicityand others.

Ang-(1-7) peptides also include other types of peptide derivativescontaining additional chemical moieties not normally part of thepeptide, provided that the derivative retains the desired functionalactivity of the peptide. Examples of such derivatives include (1) N-acylderivatives of the amino terminal or of another free amino group,wherein the acyl group may be an alkanoyl group (e.g., acetyl, hexanoyl,octanoyl) an aroyl group (e.g., benzoyl) or a blocking group such asF-moc (fluorenylmethyl-O—CO—); (2) esters of the carboxy terminal or ofanother free carboxy or hydroxyl group; (3) amide of thecarboxy-terminal or of another free carboxyl group produced by reactionwith ammonia or with a suitable amine; (4) phosphorylated derivatives;(5) derivatives conjugated to an antibody or other biological ligand andother types of derivatives; and (6) derivatives conjugated to apolyethylene glycol (PEG) chain.

Ang-(1-7) peptides may be obtained by any method of peptide synthesisknown to those skilled in the art, including synthetic (e.g., exclusivesolid phase synthesis, partial solid phase synthesis, fragmentcondensation, classical solution synthesis, native-chemical ligation)and recombinant techniques. For example, the peptides or peptidesderivatives can be obtained by solid phase peptide synthesis, which inbrief, consist of coupling the carboxyl group of the C-terminal aminoacid to a resin (e.g., benzhydrylamine resin, chloromethylated resin,hydroxymethyl resin) and successively adding N-alpha protected aminoacids. The protecting groups may be any such groups known in the art.Before each new amino acid is added to the growing chain, the protectinggroup of the previous amino acid added to the chain is removed. Suchsolid phase synthesis has been disclosed, for example, by Merrifield, J.Am. Chem. Soc. 85: 2149 (1964); Vale et al., Science 213:1394-1397(1981), in U.S. Pat. Nos. 4,305,872 and 4,316,891, Bodonsky et al. Chem.Ind. (London), 38:1597 (1966); and Pietta and Marshall, Chem. Comm. 650(1970) by techniques reviewed in Lubell et al. “Peptides” Science ofSynthesis 21.11, Chemistry of Amides. Thieme, Stuttgart, 713-809 (2005).The coupling of amino acids to appropriate resins is also well known inthe art and has been disclosed in U.S. Pat. No. 4,244,946. (Reviewed inHouver-Weyl, Methods of Organic Chemistry. Vol E22a. Synthesis ofPeptides and Peptidomimetics, Murray Goodman, Editor-in-Chief, Thieme.Stuttgart. N.Y. 2002).

Unless defined otherwise, the scientific and technological terms andnomenclature used herein have the same meaning as commonly understood bya person of ordinary skill to which this invention pertains. Generally,the procedures of cell cultures, infection, molecular biology methodsand the like are common methods used in the art. Such standardtechniques can be found in reference manuals such as, for example,Ausubel et al., Current Protocols in Molecular Biology, WileyInterscience, New York, 2001; and Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) edition, Cold Spring Harbor Laboratory Press,N.Y., 2001.

During any process of the preparation of an Ang-(1-7) peptide, it may bedesirable to protect sensitive reactive groups on any of the moleculeconcerned. This may be achieved by means of conventional protectinggroups such as those described in Protective Groups In Organic Synthesisby T. W. Greene & P. G. M. Wuts, 1991, John Wiley and Sons, New-York;and Peptides: chemistry and Biology by Sewald and Jakubke, 2002,Wiley-VCH, Wheinheim p. 142. For example, alpha amino protecting groupsinclude acyl type protecting groups (e.g., trifluoroacetyl, formyl,acetyl), aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl(BOC), cyclohexyloxycarbonyl), aromatic urethane type protecting groups(e.g., fluorenyl-9-methoxy-carbonyl (Fmoc), benzyloxycarbonyl (Cbz), Cbzderivatives) and alkyl type protecting groups (e.g., triphenyl methyl,benzyl). The amino acids side chain protecting groups include benzyl(for Thr and Ser), Cbz (Tyr, Thr, Ser, Arg, Lys), methyl ethyl,cyclohexyl (Asp, H is), Boc (Arg, His, Cys) etc. The protecting groupsmay be removed at a convenient subsequent stage using methods known inthe art.

Further, Ang-(1-7) peptides may be synthesized according to the FMOCprotocol in an organic phase with protective groups. Desirably, thepeptides are purified with a yield of 70% with high-pressure liquidchromatography (HPLC) on a C18 chromatography column and eluted with anacetonitrile gradient of 10-60%. The molecular weight of a peptide canbe verified by mass spectrometry (reviewed in Fields, G. B. “Solid-PhasePeptide Synthesis” Methods in Enzymology. Vol. 289, Academic Press,1997).

Alternatively, Ang-(1-7) peptides may be prepared in recombinant systemsusing, for example, polynucleotide sequences encoding the polypeptides.It is understood that a polypeptide may contain more than one of theabove-described modifications within the same polypeptide.

While peptides may be effective in eliciting a biological activity invitro, their effectiveness in vivo might be reduced by the presence ofproteases. Serum proteases have specific substrate requirements. Thesubstrate must have both L-amino acids and peptide bonds for cleavage.Furthermore, exopeptidases, which represent the most prominent componentof the protease activity in serum, usually act on the first peptide bondof the peptide and require a free N-terminus (Powell et al., Pharm. Res.10:1268-1273 (1993)). In light of this, it is often advantageous to usemodified versions of peptides. The modified peptides retain thestructural characteristics of the original L-amino acid peptides thatconfer the desired biological activity of Ang-(1-7) but areadvantageously not readily susceptible to cleavage by protease and/orexopeptidases.

Systematic substitution of one or more amino acids of a consensussequence with D-amino acid of the same type (e.g., D-lysine in place ofL-lysine) may be used to generate more stable peptides. Thus, a peptidederivative or peptidomimetic of the present invention may be all L, allD or mixed D, L peptide, in either forward or reverse order. Thepresence of an N-terminal or C-terminal D-amino acid increases the invivo stability of a peptide since peptidases cannot utilize a D-aminoacid as a substrate (Powell et al., Pharm. Res. 10:1268-1273 (1993)).Reverse-D peptides are peptides containing D-amino acids, arranged in areverse sequence relative to a peptide containing L-amino acids. Thus,the C-terminal residue of an L-amino acid peptide becomes N-terminal forthe D-amino acid peptide, and so forth. Reverse D-peptides retain thesame secondary conformation and therefore similar activity, as theL-amino acid peptides, but are more resistant to enzymatic degradationin vitro and in vivo, and thus can have greater therapeutic efficacythan the original peptide (Brady and Dodson, Nature 368:692-693 (1994);Jameson et al., Nature 368:744-746 (1994)). Similarly, a reverse-Lpeptide may be generated using standard methods where the C-terminus ofthe parent peptide becomes takes the place of the N-terminus of thereverse-L peptide. It is contemplated that reverse L-peptides of L-aminoacid peptides that do not have significant secondary structure (e.g.,short peptides) retain the same spacing and conformation of the sidechains of the L-amino acid peptide and therefore often have the similaractivity as the original L-amino acid peptide. Moreover, a reversepeptide may contain a combination of L- and D-amino acids. The spacingbetween amino acids and the conformation of the side chains may beretained resulting in similar activity as the original L-amino acidpeptide.

Another effective approach to confer resistance to peptidases acting onthe N-terminal or C-terminal residues of a peptide is to add chemicalgroups at the peptide termini, such that the modified peptide is nolonger a substrate for the peptidase. One such chemical modification isglycosylation of the peptides at either or both termini. Certainchemical modifications, in particular N-terminal glycosylation, havebeen shown to increase the stability of peptides in human serum (Powellet al., Pharm. Res. 10:1268-1273 (1993)). Other chemical modificationswhich enhance serum stability include, but are not limited to, theaddition of an N-terminal alkyl group, consisting of a lower alkyl offrom one to twenty carbons, such as an acetyl group, and/or the additionof a C-terminal amide or substituted amide group. In particular, thepresent invention includes modified peptides consisting of peptidesbearing an N-terminal acetyl group and/or a C-terminal amide group.

Substitution of non-naturally-occurring amino acids for natural aminoacids in a subsequence of the peptides can also confer resistance toproteolysis. Such a substitution can, for instance, confer resistance toproteolysis by exopeptidases acting on the N-terminus without affectingbiological activity. Examples of non-naturally-occurring amino acidsinclude α,α-disubstituted amino acids, N-alkyl amino acids, C-α-methylamino acids, β-amino acids, and β-methyl amino acids. Amino acidsanalogs useful in the present invention may include, but are not limitedto, β-alanine, norvaline, norleucine, 4-aminobutyric acid, orithine,hydroxyproline, sarcosine, citrulline, cysteic acid, cyclohexylalanine,2-aminoisobutyric acid, 6-aminohexanoic acid, t-butylglycine,phenylglycine, o-phosphoserine, N-acetyl serine, N-formylmethionine,3-methylhistidine and other unconventional amino acids. Furthermore, thesynthesis of peptides with non-naturally-occurring amino acids isroutine in the art.

In addition, constrained peptides comprising a consensus sequence or asubstantially identical consensus sequence variation may be generated bymethods well known in the art (Rizo and Gierasch, Ann. Rev. Biochem.61:387-418 (1992)). For example, constrained peptides may be generatedby adding cysteine residues capable of forming disulfide bridges and,thereby, resulting in a cyclic peptide. Cyclic peptides can beconstructed to have no free N- or C-termini. Accordingly, they are notsusceptible to proteolysis by exopeptidases, although they may besusceptible to endopeptidases, which do not cleave at peptide termini.The amino acid sequences of the peptides with N-terminal or C-terminalD-amino acids and of the cyclic peptides are usually identical to thesequences of the peptides to which they correspond, except for thepresence of N-terminal or C-terminal D-amino acid residue, or theircircular structure, respectively.

Cyclic Peptides

In some embodiments, a functional equivalent, analogue or derivative ofnaturally-occurring Ang-(1-7) is a cyclic peptide. As used herein, acyclic peptide has an intramolecular covalent bond between twonon-adjacent residues. The intramolecular bond may be a backbone tobackbone, side-chain to backbone or side-chain to side-chain bond (i.e.,terminal functional groups of a linear peptide and/or side-chainfunctional groups of a terminal or interior residue may be linked toachieve cyclization). Typical intramolecular bonds include disulfide,amide and thioether bonds. A variety of means for cyclizing polypeptidesare well known in the art, as are many other modifications that can bemade to such peptides. For a general discussion, see InternationalPatent Publication Nos. WO 01/53331 and WO 98/02452, the contents ofwhich are incorporated herein by reference. Such cyclic bonds and othermodifications can also be applied to the cyclic peptides and derivativecompounds of this invention.

Cyclic peptides as described herein may comprise residues of L-aminoacids, D-amino acids, or any combination thereof. Amino acids may befrom natural or non-natural sources, provided that at least one aminogroup and at least one carboxyl group are present in the molecule; α-and β-amino acids are generally preferred. Cyclic peptides may alsocontain one or more rare amino acids (such as 4-hydroxyproline orhydroxylysine), organic acids or amides and/or derivatives of commonamino acids, such as amino acids having the C-terminal carboxylateesterified (e.g., benzyl, methyl or ethyl ester) or amidated and/orhaving modifications of the N-terminal amino group (e.g., acetylation oralkoxycarbonylation), with or without any of a wide variety ofside-chain modifications and/or substitutions (e.g., methylation,benzylation, t-butylation, tosylation, alkoxycarbonylation, and thelike). Suitable derivatives include amino acids having an N-acetyl group(such that the amino group that represents the N-terminus of the linearpeptide prior to cyclization is acetylated) and/or a C-terminal amidegroup (i.e., the carboxy terminus of the linear peptide prior tocyclization is amidated). Residues other than common amino acids thatmay be present with a cyclic peptide include, but are not limited to,penicillamine, β,β-tetramethylene cysteine, β,β-pentamethylene cysteine,β-mercaptopropionic acid, β,β-pentamethylene-β-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, α-aminoadipic acid, m-aminomethylbenzoic acid andα,β-diaminopropionic acid.

Following synthesis of a linear peptide, with or without N-acetylationand/or C-amidation, cyclization may be achieved by any of a variety oftechniques well known in the art. Within one embodiment, a bond may begenerated between reactive amino acid side chains. For example, adisulfide bridge may be formed from a linear peptide comprising twothiol-containing residues by oxidizing the peptide using any of avariety of methods. Within one such method, air oxidation of thiols cangenerate disulfide linkages over a period of several days using eitherbasic or neutral aqueous media. The peptide is used in high dilution tominimize aggregation and intermolecular side reactions. Alternatively,strong oxidizing agents such as I₂ and K₃Fe(CN)₆ can be used to formdisulfide linkages. Those of ordinary skill in the art will recognizethat care must be taken not to oxidize the sensitive side chains of Met,Tyr, Trp or His. Within further embodiments, cyclization may be achievedby amide bond formation. For example, a peptide bond may be formedbetween terminal functional groups (i.e., the amino and carboxy terminiof a linear peptide prior to cyclization). Within another suchembodiment, the linear peptide comprises a D-amino acid. Alternatively,cyclization may be accomplished by linking one terminus and a residueside chain or using two side chains, with or without an N-terminalacetyl group and/or a C-terminal amide. Residues capable of forming alactam bond include lysine, ornithine (Orn), α-amino adipic acid,m-aminomethylbenzoic acid, α,β-diaminopropionic acid, glutamate oraspartate. Methods for forming amide bonds are generally well known inthe art. Within one such method, carbodiimide-mediated lactam formationcan be accomplished by reaction of the carboxylic acid with DCC, DIC, EDAC or DCCI, resulting in the formation of an O-acylurea that can bereacted immediately with the free amino group to complete thecyclization. Alternatively, cyclization can be performed using the azidemethod, in which a reactive azide intermediate is generated from analkyl ester via a hydrazide. Alternatively, cyclization can beaccomplished using activated esters. The presence of electronwithdrawing substituents on the alkoxy carbon of esters increases theirsusceptibility to aminolysis. The high reactivity of esters ofp-nitrophenol, N-hydroxy compounds and polyhalogenated phenols has madethese “active esters” useful in the synthesis of amide bonds. Within afurther embodiment, a thioether linkage may be formed between the sidechain of a thiol-containing residue and an appropriately derivatizedα-amino acid. By way of example, a lysine side chain can be coupled tobromoacetic acid through the carbodiimide coupling method (DCC, EDAC)and then reacted with the side chain of any of the thiol containingresidues mentioned above to form a thioether linkage. In order to formdithioethers, any two thiol containing side-chains can be reacted withdibromoethane and diisopropylamine in DMF.

Exemplary Angiotensin-(1-7) Peptides

In certain aspects, the invention provides linear angiotensin-(1-7)peptides. As discussed above, the structure of naturally-occurringAng-(1-7) is as follows:

Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO: 1)

The peptides and peptide analogs of the invention can be generallyrepresented by the following sequence:

Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷, (SEQ ID NO: 4)or a pharmaceutically acceptable salt thereof.

Xaa¹ is any amino acid or a dicarboxylic acid. In certain embodiments,Xaa¹ is Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala,Me₂Gly (N,N-dimethylglycine), Pro, Bet (betaine,1-carboxy-N,N,N-trimethylmethanaminium hydroxide), Glu, Gly, Asp, Sar(sarcosine) or Suc (succinic acid). In certain such embodiments, Xaa¹ isa negatively-charged amino acid, such as Asp or Glu, typically Asp.

Xaa² is Arg, Lys, Ala, Cit (citrulline), Orn (ornithine), acetylatedSer, Sar, D-Arg and D-Lys. In certain embodiments, Xaa² is apositively-charged amino acid such as Arg or Lys, typically Arg.

Xaa³ is Val, Ala, Leu, Nle (norleucine), Ile, Gly, Lys, Pro, HydroxyPro(hydroxyproline), Aib (2-aminoisobutyric acid), Acpc or Tyr. In certainembodiments, Xaa³ is an aliphatic amino acid such as Val, Leu, Ile orNle, typically Val or Nle.

Xaa⁴ is Tyr, Tyr(PO₃), Thr, Ser, homoSer (homoserine), azaTyr(aza-α¹-homo-L-tyrosine) or Ala. In certain embodiments, Xaa⁴ is ahydroxyl-substituted amino acid such as Tyr, Ser or Thr, typically Tyr.

Xaa⁵ is Ile, Ala, Leu, norLeu, Val or Gly. In certain embodiments, Xaa⁵is an aliphatic amino acid such as Val, Leu, Ile or Nle, typically Ile.

Xaa⁶ is His, Arg or 6-NH₂-Phe (6-aminophenylalaine). In certainembodiments, Xaa⁶ is a fully or partially positively-charged amino acidsuch as Arg or His.

Xaa⁷ is Cys, Pro or Ala.

In certain embodiments, one or more of Xaa¹-Xaa⁷ is identical to thecorresponding amino acid in naturally-occurring Ang-(1-7). In certainsuch embodiments, all but one or two of Xaa¹-Xaa¹ are identical to thecorresponding amino acid in naturally-occurring Ang-(1-7). In otherembodiments, all of Xaa¹-Xaa⁶ are identical to the corresponding aminoacid in naturally-occurring Ang-(1-7).

In certain embodiments, Xaa³ is Nle. When Xaa³ is Nle, one or more ofXaa¹-Xaa² and Xaa⁴⁻⁷ are optionally identical to the corresponding aminoacid in naturally-occurring Ang-(1-7). In certain such embodiments, allbut one or two of Xaa¹-Xaa² and Xaa⁴⁻⁷ are identical to thecorresponding amino acid in naturally-occurring Ang-(1-7). In otherembodiments, all of Xaa¹-Xaa² and Xaa⁴⁻⁷ are identical to thecorresponding amino acid in naturally-occurring Ang-(1-7), resulting inthe amino acid sequence: Asp¹-Arg²-Nle³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ IDNO:5).

In certain embodiments, the peptide has the amino acid sequenceAsp¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO:6) orAsp¹-Arg²-Val³-ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO:2).

Exemplary Cyclic Angiotensin (1-7) Peptides

In certain aspects, the invention provides a cyclic angiotensin-(1-7)(Ang-(1-7)) peptide analog comprising a linkage, such as between theside chains of amino acids corresponding to positions Tyr⁴ and Pro⁷ inAng. These peptide analogs typically comprise 7 amino acid residues, butcan also include a cleavable sequence. As discussed in greater detailbelow, the invention includes fragments and analogs where one or moreamino acids are substituted by another amino acid (including fragments).One example of such an analog is Asp¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQID NO: 22), wherein a linkage is formed between Ser⁴ and Cys⁷.

Although the following section describes aspects of the invention interms of a thioether bond linking residues at the 4- and 7-positions, itshould be understood that other linkages (as described above) couldreplace the thioether bridge and that other residues could be cyclized.A thioether bridge is also referred to as a monosulfide bridge or, inthe case of Ala-S-Ala, as a lanthionine bridge. Thioetherbridge-containing peptides can be formed by two amino acids having oneof the following formulas:

In these formulae, R¹, R², R³, R⁴, R⁵ and R⁶ are independently —H, analkyl (e.g., C₁-C₆ alkyl, C₁-C₄ alkyl) or an aralkyl group, where thealkyl and aralkyl groups are optionally substituted with one or morehalogen, —OH or —NRR′ groups (where R and R′ are independently —H orC₁-C₄ alkyl). In certain embodiments, R¹, R², R³, R⁴, R⁵ and R⁶ are eachindependently —H or —CH₃, such where all are —H.

In certain embodiments, the invention provides an Ang analog orderivative comprising a thioether bridge according to formula (I).Typically, R¹, R², R³ and R⁴ are independently selected from —H and—CH₃. Peptides comprising a thioether bridge according to formula (I)can be produced, for example, by lantibiotic enzymes or by sulfurextrusion of a disulfide. In one example, the disulfide from which thesulfur is extruded can be formed by D-cysteine in position 4 andL-cysteine in position 7 or by D-cysteine in position 4 andL-penicillamine in position 7 (see, e.g., Galande, Trent and Spatola(2003) Biopolymers 71, 534-551).

In other embodiments, the linkage of the two amino acids can be thebridges depicted in Formula (II) or Formula (III). Peptides comprising athioether bridge according to Formula (II) can be made, for example, bysulfur extrusion of a disulfide formed by D-homocysteine in position 4and L-cysteine in position 7. Similarly, peptides comprising a thioetherbridge as in Formula (III) can be made, for example, by sulfur extrusionof a disulfide formed by D-cysteine in position 4 and L-homocysteine inposition 7.

As discussed above, the Ang analogs and derivatives of the inventionvary in length and amino acid composition. The Ang analogs andderivatives of the invention preferably have biological activity or arean inactive precursor molecule that can be proteolytically activated(such as how angiotensin(I), with 10 amino acids, is converted to activefragments by cleavage of 2 amino acids). The size of an Ang analog orderivative can vary but is typically between from about 5 to 10 aminoacids, as long as the “core” pentameric segment comprising the 3-7Nle-thioether-ring structure is encompassed. The amino acid sequence ofan analog or derivative of the invention can vary, typically providedthat it is biologically active or can become proteolytically activated.Biological activity of an analog or derivative can be determined usingmethods known in the art, including radioligand binding studies, invitro cell activation assays and in vivo experiments. See, for example,Godeny and Sayeski, (2006) Am. J. Physiol. Cell. Physiol.291:C1297-1307; Sarr et al., Cardiovasc. Res. (2006) 71:794-802; andKoziarz et al., (1933) Gen. Pharmacol. 24:705-713.

Ang analogs and derivatives where only the length of the peptide isvaried include the following:

a 4,7-cyclized analog designated [Cyc⁴⁻⁷]Ang-(1-7), which is derivedfrom natural Ang-(1-7) (Asp¹-Arg²-Val³-Cyc⁴-Ile⁵-His⁶-Cyc⁷, SEQ IDNO:7).

a 4,7-cyclized analog designated [Nle³, Cyc⁴⁻⁷]Ang-(1-10), which isderived from natural Angiotensin I (Ang-(1-10))(Asp¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸-His⁹-Leu¹⁰, SEQ ID NO:8);

a 4,7-cyclized analog designated [Nle³, Cyc⁴⁻⁷]Ang-(1-8), which isderived from natural Angiotensin II (Ang-(1-8))(Asp¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸, SEQ ID NO:9);

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷]Ang-(2-8), which isderived from natural Angiotensin III (Ang-(2-8))(Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸, SEQ ID NO:10);

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷]Ang-(3-8), which isderived from natural Angiotensin IV (Ang-(3-8))(Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸, SEQ ID NO:11);

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷]Ang-(1-7) derived fromnatural Ang-(1-7) (Asp¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷, SEQ ID NO:12);and

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷]Ang-(1-9) derived fromnatural Ang-(1-9) (Asp¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸-His⁹, SEQ IDNO:13).

These analogs can have one of the thioether bridges shown in Formulae(I)-(III) as the Cyc⁴⁻⁷ moiety, for example, where Cyc⁴ and Cyc⁷ arerepresented by Formula (I), such as where R¹-R⁴ are each —H or —CH₃,typically —H.

As compared to the amino acid sequence of the natural angiotensinpeptide, the amino acids at positions 4 and 7 of the Cyc⁴⁻⁷ analog aremodified to allow introduction of the thioether-ring structures shownabove. In addition to the length of the Ang analogs, the amino acids atpositions other than 3, 4 and 7 can be the same or different from thenaturally-occurring peptide, typically provided that the analog retainsa biological function. For analogs of inactive precursors, like[Cyc⁴⁻⁷]Ang-(1-10), biological function refers to one or both of ananalog's susceptibility to angiotensin-converting enzymes that cancleave it to a biologically active fragment (e.g. Ang-(1-8) orAng-(1-7)) or the biological activity of the fragment itself. In certainembodiments, an Ang analog or derivative of the invention has nointrinsic function but inhibits the effects of one or morenaturally-occurring angiotensin compounds.

In certain embodiments, an Ang analog of the invention is represented byFormula (IV):

Xaa¹-Xaa²-Xaa³-Cyc⁴-Xaa⁵-Xaa⁶-Cyc⁷ (IV, SEQ ID NO: 14)

Xaa¹ is any amino acid, but typically a negatively-charged amino acidsuch as Glu or Asp, more typically Asp.

Xaa² is a positively-charged amino acid such as Arg or Lys, typicallyArg.

Xaa³ is an aliphatic amino acid, such as Leu, Ile or Val, typically Val.

Cyc⁴ forms a thioether bridge in conjunction with Cyc⁷. Cyc⁴ can be aD-stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer.Examples of Cyc⁴ (taken with Cyc⁷) are shown in Formulas (I), (II) and(III). Typically, the R groups in Formulae (I), (II) and (III) are —H or—CH₃, especially —H.

Xaa⁵ is an aliphatic amino acid, such as Leu, Ile or Val, typically Ile.

Xaa⁶ is His.

Cyc⁷ forms a thioether bridge in conjunction with Cyc⁴, such as inFormula (I), (II) or (III). Cyc⁷ can be a D-stereoisomer and/or aL-stereoisomer, typically a L-stereoisomer. Examples of Cyc⁷ (taken withCyc⁴) are shown in Formulas (I), (II), (III) and (IV). Typically, the Rgroups in Formulae Formulas (I), (II),) and (III) and (IV) are —H or—CH₃, especially —H.

In certain embodiments, one or more of Xaa¹-Xaa⁶ (excluding Cyc⁴ andCyc⁷) is identical to the corresponding amino acid innaturally-occurring Ang-(1-7). In certain such embodiments, all but oneor two of Xaa¹-Xaa⁶ are identical to the corresponding amino acid innaturally-occurring Ang-(1-7). In other embodiments, all of Xaa¹-Xaa⁶are identical to the corresponding amino acid in naturally-occurringAng-(1-7).

In certain embodiments, Cyc⁴ and Cyc⁷ are independently selected fromAbu (2-aminobutyric acid) and Ala (alanine), where Ala is present in atleast one position. Thus, cyclic analogs can have a thioether linkageformed by -Ala⁴-S-Ala⁷-(Formula (I), where R¹-R⁴ are each —H);-Ala⁴-S-Abu⁷-(Formula (I): R¹-R³ are —H and R⁴ is —CH₃) or-Abu⁴-S-Ala⁷-(Formula (I): R¹, R³ and R⁴ are —H and R² is —CH₃).Specific examples of cyclic analogs comprise a -Abu⁴-S-Ala⁷- or-Ala⁴-S-Ala⁷-linkage.

In certain embodiments, the invention provides an Ang-(1-7) analog witha thioether-bridge between position 4 and position 7 having the aminoacid sequence Asp¹-Arg²-Val³-Abu⁴-Ile⁵-His⁶-Ala⁷ (SEQ ID NO:15) or theamino acid sequence Asp¹-Arg²-Val³-Ala⁴-Ile⁵-His⁶-Ala⁷ (SEQ ID NO:16),which are represented by the following structural diagrams:

In certain embodiments, an Ang analog or derivative of the invention isrepresented by Formula (V):

Xaa¹-Xaa²-Nle³-Cyc⁴-Xaa⁵-Xaa⁶-Cyc⁷-Xaa⁸-Xaa⁹-Xaa¹⁰ (V, SEQ ID NO:17) Asdiscussed above, one or more of Xaa¹, Xaa², Xaa⁸, Xaa⁹ and Xaa¹⁰ areabsent in certain embodiments. For example, (1) Xaa¹⁰ is absent, (2)Xaa⁹ and Xaa¹⁰ are absent, (3) Xaa⁸, Xaa⁹ and Xaa¹⁰ are absent, (4) Xaa¹is absent, (5) Xaa¹ and Xaa¹⁰ are absent, (6) Xaa¹, Xaa⁹ and Xaa¹⁰ areabsent, (7) Xaa¹, Xaa⁸, Xaa⁹ and Xaa¹⁰ are absent, (8) Xaa¹ and Xaa² areabsent, (9) Xaa¹, Xaa² and Xaa¹⁰ are absent, (10) Xaa¹, Xaa², Xaa⁹ andXaa¹⁰ are absent, or (11) Xaa¹, Xaa², Xaa⁸, Xaa⁹ and Xaa¹⁰ are absent.For each of these embodiments, the remaining amino acids have the valuesdescribed below.

Xaa¹, when present, is any amino acid, but typically a negativelycharged amino acid such as Glu or Asp, more typically Asp.

Xaa², when present, is a positively charged amino acid such as Arg orLys, typically Arg.

Nle³ is norleucine.

Cyc⁴ forms a thioether bridge in conjunction with Cyc⁷. Cyc⁴ can be aD-stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer.Examples of Cyc⁴ (taken with Cyc⁷) are shown in Formulas (I), (II) and(III). Typically, the R groups in Formulae (I), (II) and (III) are —H or—CH₃, especially —H.

Xaa⁵ is an aliphatic amino acid, such as Leu, Nle, Ile or Val, typicallyIle.

Xaa⁶ is His.

Cyc⁷ forms a thioether bridge in conjunction with Cyc⁴, such as inFormula (I), (II) or (III). Cyc⁷ can be a D-stereoisomer and/or aL-stereoisomer, typically a L-stereoisomer. Examples of Cyc⁷ (taken withCyc⁴) are shown in Formulas (I), (II) and (III). Typically, the R groupsin Formulae (I), (II) and (III) are —H or —CH₃, especially —H.

Xaa⁸, when present, is an amino acid other than Pro, typically Phe orIle. In certain embodiments, Ile results in an inhibitor of Ang(1-8). Incertain embodiments, Phe maintains the biological activity of Ang(1-8)or Ang(1-10).

Xaa⁹, when present, is His.

Xaa¹⁰, when present, is an aliphatic residue, for example, Ile, Val orLeu, typically Leu.

In certain embodiments, one or more of Xaa¹-Xaa¹⁰ (excluding Nle³, Cyc⁴and Cyc⁷) is identical to the corresponding amino acid innaturally-occurring Ang (including Ang-(1-7), Ang(1-8), Ang(1-9),Ang(1-10), Ang(2-7), Ang(2-8), Ang(2-9), Ang(2-10), Ang(3-8), Ang(3-9)and Ang(3-10). In certain such embodiments, all but one or two ofXaa¹-Xaa¹⁰ (for those present) are identical to the corresponding aminoacid in naturally-occurring Ang. In other embodiments, all of Xaa¹-Xaa¹⁰(for those present) are identical to the corresponding amino acid innaturally-occurring Ang.

In certain embodiments, Cyc⁴ and Cyc⁷ are independently selected fromAbu (2-aminobutyric acid) and Ala (alanine), where Ala is present at atleast one position. Thus, encompassed are cyclic analogs comprising athioether linkage formed by -Ala⁴-S-Ala⁷-(Formula (I), where R¹-R⁴ areeach —H); -Ala⁴-S-Abu⁷-(Formula (I): R¹-R³ are —H and R⁴ is —CH₃) or-Abu⁴-S-Ala⁷-(Formula (I): R¹, R³ and R⁴ are —H and R² is —CH₃).Specific cyclic analogs comprise a -Abu⁴-S-Ala⁷- or-Ala⁴-S-Ala⁷-linkage.

In particular, the invention provides an Ang-(1-7) analog or derivativewith a thioether-bridge between position 4 and position 7 having theamino acid sequence Asp¹-Arg²-Nle³-Abu⁴-Ile⁵-His⁶-Ala⁷ (SEQ ID NO:18) orthe amino acid sequence Asp¹-Arg²-Nle³-Ala⁴-Ile⁵-His⁶-Ala⁷ (SEQ IDNO:19).

In another aspect, the invention provides an Ang-(1-8) analog orderivative with a thioether-bridge between position 4 and position 7having Ang-(1-8) antagonistic activity, in particular an Ang(1-8) analogor derivative having the amino acid sequenceAsp¹-Arg²-Nle³-Abu⁴-Ile⁵-His⁶-Ala⁷-Ile⁸ (SEQ ID NO:20), or the aminoacid sequence Asp¹-Arg²-Nle³-Ala⁴-Ile⁵-His⁶-Ala⁷-Ile⁸ (SEQ ID NO:21).

An alkyl group is a straight chained or branched non-aromatichydrocarbon that is completely saturated. Typically, a straight chainedor branched alkyl group has from 1 to about 20 carbon atoms, preferablyfrom 1 to about 10. Examples of straight chained and branched alkylgroups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,tert-butyl, pentyl, hexyl, pentyl and octyl. A C1-C4 straight chained orbranched alkyl group is also referred to as a “lower alkyl” group.

An aralkyl group is an alkyl group substituted by an aryl group.Aromatic (aryl) groups include carbocyclic aromatic groups such asphenyl, naphthyl, and anthracyl, and heteroaryl groups such asimidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl,pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl. Aromaticgroups also include fused polycyclic aromatic ring systems in which acarbocyclic aromatic ring or heteroaryl ring is fused to one or moreother heteroaryl rings. Examples include benzothienyl, benzofuryl,indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole,quinolinyl, isoquinolinyl and isoindolyl.

Ang (1-7) Receptor Agonists

In some embodiments, the present invention provides methods of treatingbrain conditions including administering to a subject who is sufferingfrom or susceptible to one or more brain conditions an angiotensin (1-7)receptor agonist. As used herein, the term “angiotensin-(1-7) receptoragonist” encompasses any molecule that has a positive impact in afunction of an angiotensin-(1-7) receptor, in particular, the G-proteincoupled Mas receptor. In some embodiments, an angiotensin-(1-7) receptoragonist directly or indirectly enhances, strengthens, activates and/orincreases an angiotensin-(1-7) receptor (i.e., the Mas receptor)activity. In some embodiments, an angiotensin-(1-7) receptor agonistdirectly interacts with an angiotensin-(1-7) receptor (i.e., the Masreceptor). Such agonists can be peptidic or non-peptidic including,e.g., proteins, chemical compounds, small molecules, nucleic acids,antibodies, drugs, ligands, or other agents. In some embodiments, theangiotensin (1-7) receptor agonist is a non-peptidic agonist.

An exemplary class of angiotensin-(1-7) receptor agonists are1-(p-thienylbenzyl)imidazoles. Examples of these non-peptideangiotensin-(1-7) receptor agonists are represented by StructuralFormula (VI):

or pharmaceutically acceptable salts thereof, wherein:

R¹ is halogen, hydroxyl, (C₁-C₄)-alkoxy, (C₁-C₈)-alkoxy wherein 1 to 6carbon atoms are replaced by the heteroatoms O, S, or NH (preferably byO), (C₁-C₄)-alkoxy substituted by a saturated cyclic ether such astetrahydropyran or tetrahydrofuran, O—(C₁-C₄)-alkenyl,O—(C₁-C₄)—alkylaryl, or aryloxy that is unsubstituted or substituted bya substituent selected from halogen, (C₁-C₃)-alkyl, (C₁-C₃)-alkoxy andtrifluoromethyl;

R² is CHO, COOH, or (3) CO—O—(C₁-C₄)-alkyl;

R³ is (C₁-C₄)-alkyl or aryl;

R⁴ is hydrogen, halogen (chloro, bromo, fluoro), or (C₁-C₄)-alkyl;

X is oxygen or sulfur;

Y is oxygen or —NH—;

R⁵ is hydrogen, (C₁-C₆)-alkyl; or (C₁-C₄)-alkylaryl, where R⁵ ishydrogen when Y is —NH—; and

R⁶ is (C₁-C₅)-alkyl.

In certain embodiments, R¹ is not halogen when R² is COOH orCO—O—(C₁-C₄)-alkyl.

In some embodiments, an angiotensin-(1-7) receptor agonist is AVE 0991,5-formyl-4-methoxy-2-phenyl-1[[4-[2-(ethylaminocarbonylsulfonamido)-5-isobutyl-3-thienyl]-phenyl]-methyl]-imidazole,which is represented by the following structure:

Another exemplary class of angiotensin-(1-7) receptor agonists arep-thienylbenzylamides. Examples of these non-peptide angiotensin-(1-7)receptor agonists are represented by Structural Formula (VII):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is (C₁-C₅)-alkyl that is unsubstituted or substituted by a radicalchosen from NH₂, halogen, O—(C₁-C₃)-alkyl, CO—O—(C₁-C₃)-alkyl and CO₂H,(C₃-C₈)-cycloalkyl, (C₁-C₃)-alkyl-(C₃-C₈)-cycloalkyl, (C₆-C₁₀)-aryl thatis unsubstituted or substituted by a radical chosen from halogen andO—(C₁-C₃)-alkyl, (C₁-C₃)-alkyl-(C₆-C₁₀)-aryl where the aryl radical isunsubstituted or substituted by a radical chosen from halogen andO—(C₁-C₃)-alkyl, (C₁-C₅)-heteroaryl, or(C₁-C₃)-alkyl-(C₁-C₅)-heteroaryl;

R² is hydrogen, (C₁-C₆)-alkyl that is unsubstituted or substituted by aradical chosen from halogen and O—(C₁-C₃)-alkyl, (C₃-C₈)-cycloalkyl,(C₁-C₃)-alkyl-(C₃-C₈)-cycloalkyl, (C₆-C₁₀)-aryl that is unsubstituted orsubstituted by a radical chosen from among halogen, O—(C₁-C₃)-alkyl andCO—O—(C₁-C₃)-alkyl, or (C₁-C₃)-alkyl-(C₆-C₁₀)-aryl that is unsubstitutedor substituted by a radical chosen from halogen and O—(C₁-C₃)-alkyl;

R³ is hydrogen, COOH, or COO—(C₁-C₄)-alkyl;

R⁴ is hydrogen, halogen; or (C₁-C₄)-alkyl;

R⁵ is hydrogen or (C₁-C₆)-alkyl;

R⁶ is hydrogen, (C₁-C₆)-alkyl, (C₁-C₃)-alkyl-(C₃-C₈)-cycloalkyl, or(C₂-C₆)-alkenyl; and

X is oxygen or NH.

Additional examples of angiotensin-(1-7) receptor agonists are describedin U.S. Pat. No. 6,235,766, the contents of which are incorporated byreference herein.

Various angiotensin-(1-7) receptor agonists described above can bepresent as pharmaceutically acceptable salts. As used herein, “apharmaceutically acceptable salt” refers to salts that retain thedesired activity of the peptide or equivalent compound, but preferablydo not detrimentally affect the activity of the peptide or othercomponent of a system, which uses the peptide. Examples of such saltsare acid addition salts formed with inorganic acids, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid, and the like. Salts may also be formed with organic acidssuch as, for example, acetic acid, oxalic acid, tartaric acid, succinicacid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, and the like. Salts formed from a cationic materialmay utilize the conjugate base of these inorganic and organic acids.Salts may also be formed with polyvalent metal cations such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickeland the like or with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine, or combinations thereof(e.g., a zinc tannate salt). The non-toxic, physiologically acceptablesalts are preferred.

The salts can be formed by conventional means such as by reacting thefree acid or free base forms of the product with one or more equivalentsof the appropriate acid or base in a solvent or medium in which the saltis insoluble, or in a solvent such as water which is then removed invacuo or by freeze-drying, or by exchanging the cations of an existingsalt for another cation on a suitable ion exchange resin.

An alkyl group is a straight chained or branched non-aromatichydrocarbon that is completely saturated. Typically, a straight chainedor branched alkyl group has from 1 to about 20 carbon atoms, preferablyfrom 1 to about 10. Examples of straight chained and branched alkylgroups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,tert-butyl, pentyl, hexyl, pentyl and octyl. A C1-C4 straight chained orbranched alkyl group is also referred to as a “lower alkyl” group.

An alkenyl group is a straight chained or branched non-aromatichydrocarbon that is includes one or more double bonds. Typically, astraight chained or branched alkenyl group has from 2 to about 20 carbonatoms, preferably from 2 to about 10. Examples of straight chained andbranched alkenyl groups include ethenyl, n-propenyl, and n-butenyl.

Aromatic (aryl) groups include carbocyclic aromatic groups such asphenyl, naphthyl, and anthracyl, and heteroaryl groups such asimidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl,pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl. Aromaticgroups also include fused polycyclic aromatic ring systems in which acarbocyclic aromatic ring or heteroaryl ring is fused to one or moreother heteroaryl rings. Examples include benzothienyl, benzofuryl,indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole,quinolinyl, isoquinolinyl and isoindolyl.

An aralkyl group is an alkyl group substituted by an aryl group.

Formulations

In accordance with the methods of the invention, an Ang (1-7) peptide orangiotensin (1-7) receptor agonist as described herein of the inventioncan be administered to a subject alone (e.g., as a purified peptide orcompound), or as a component of a composition or medicament (e.g., inthe manufacture of a medicament for the treatment of the disease), asdescribed herein. The compositions can be formulated with aphysiologically acceptable carrier or excipient to prepare apharmaceutical composition. The carrier and composition can be sterile.The formulation should suit the mode of administration, for exampleintravenous or subcutaneous administration. Methods of formulatingcompositions are known in the art (see, e.g., Remington'sPharmaceuticals Sciences, 17^(th) Edition, Mack Publishing Co., (AlfonsoR. Gennaro, editor) (1989)).

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, sugars such as mannitol, sucrose, or others,dextrose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc., as well as combinations thereof. The pharmaceuticalpreparations can, if desired, be mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, coloring and/oraromatic substances and the like) which do not deleteriously react withthe active compounds or interference with their activity. In a preferredembodiment, a water-soluble carrier suitable for intravenousadministration is used.

The composition or medicament, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, sustainedrelease formulation, or powder. The composition can also be formulatedas a suppository, with traditional binders and carriers such astriglycerides.

The composition or medicament can be formulated in accordance with theroutine procedures as a pharmaceutical composition adapted foradministration to human beings. For example, in a preferred embodiment,a composition for intravenous administration typically is a solution insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic to ease pain atthe site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water, saline or dextrose/water. Where thecomposition is administered by injection, an ampule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

An Ang (1-7) peptide or angiotensin (1-7) receptor agonist as describedherein can be formulated as neutral or salt forms. Pharmaceuticallyacceptable salts include those formed with free amino groups such asthose derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, etc., and those formed with free carboxyl groups such as thosederived from sodium, potassium, ammonium, calcium, ferric hydroxides,isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,procaine, etc.

An Ang (1-7) peptide or angiotensin (1-7) receptor agonist as describedherein (or a composition or medicament containing an Ang (1-7) peptideor angiotensin (1-7) receptor agonist described herein) is administeredby any appropriate route. In some embodiments, an Ang (1-7) peptide orangiotensin (1-7) receptor agonist described herein is administeredsubcutaneously. As used herein, the term “subcutaneous tissue”, isdefined as a layer of loose, irregular connective tissue immediatelybeneath the skin. For example, the subcutaneous administration may beperformed by injecting a composition into areas including, but notlimited to, thigh region, abdominal region, gluteal region, or scapularregion. In some embodiments, an Ang (1-7) peptide or angiotensin (1-7)receptor agonist described herein is administered intravenously.Alternatively, an Ang (1-7) peptide or angiotensin (1-7) receptoragonist described herein (or a composition or medicament containing anAng (1-7) peptide or angiotensin (1-7) receptor agonist describedherein) can be administered by inhalation, parenterally, intradermally,transdermally, rectally, or transmucosally. More than one route can beused concurrently, if desired.

In some embodiments, a composition is administered in a therapeuticallyeffective amount and/or according to a dosing regimen that is correlatedwith a particular desired outcome (e.g., with treating or reducing riskfor ischemic stroke).

Particular doses or amounts to be administered in accordance with thepresent invention may vary, for example, depending on the nature and/orextent of the desired outcome, on particulars of route and/or timing ofadministration, and/or on one or more characteristics (e.g., weight,age, personal history, genetic characteristic, lifestyle parameter,severity of cardiac defect and/or level of risk of cardiac defect, etc.,or combinations thereof). Such doses or amounts can be determined bythose of ordinary skill. In some embodiments, an appropriate dose oramount is determined in accordance with standard clinical techniques.For example, in some embodiments, an appropriate dose or amount is adose or amount sufficient to reduce a disease severity index score by 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more.For example, in some embodiments, an appropriate dose or amount is adose or amount sufficient to reduce a disease severity index score by 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%.Alternatively or additionally, in some embodiments, an appropriate doseor amount is determined through use of one or more in vitro or in vivoassays to help identify desirable or optimal dosage ranges or amounts tobe administered.

In various embodiments, an Ang (1-7) peptide or angiotensin (1-7)receptor agonist is administered at a therapeutically effective amount.As used herein, the term “therapeutically effective amount” is largelydetermined based on the total amount of the therapeutic agent containedin the pharmaceutical compositions of the present invention. Generally,a therapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating the underlying disease or condition). In someparticular embodiments, appropriate doses or amounts to be administeredmay be extrapolated from dose-response curves derived from in vitro oranimal model test systems.

Therapeutically effective dosage amounts of angiotensin (1-7) peptidesor angiotensin (1-7) receptor agonists, including derivatives, analogs,and/or salts may be present in varying amounts in various embodiments.For example, in some embodiments, a therapeutically effective amount ofan angiotensin (1-7) peptide may be an amount ranging from about 10-1000mg (e.g., about 20 mg-1,000 mg, 30 mg-1,000 mg, 40 mg-1,000 mg, 50mg-1,000 mg, 60 mg-1,000 mg, 70 mg-1,000 mg, 80 mg-1,000 mg, 90 mg-1,000mg, about 10-900 mg, 10-800 mg, 10-700 mg, 10-600 mg, 10-500 mg,100-1000 mg, 100-900 mg, 100-800 mg, 100-700 mg, 100-600 mg, 100-500 mg,100-400 mg, 100-300 mg, 200-1000 mg, 200-900 mg, 200-800 mg, 200-700 mg,200-600 mg, 200-500 mg, 200-400 mg, 300-1000 mg, 300-900 mg, 300-800 mg,300-700 mg, 300-600 mg, 300-500 mg, 400 mg-1,000 mg, 500 mg-1,000 mg,100 mg-900 mg, 200 mg-800 mg, 300 mg-700 mg, 400 mg-700 mg, and 500mg-600 mg). In some embodiments, an angiotensin (1-7) peptide orangiotensin (1-7) receptor agonist is present in an amount of or greaterthan about 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg,400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg.In some embodiments, an angiotensin (1-7) peptide or angiotensin (1-7)receptor agonist is present in an amount of or less than about 1000 mg,950 mg, 900 mg, 850 mg, 800 mg, 750 mg, 700 mg, 650 mg, 600 mg, 550 mg,500 mg, 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, or 100mg. In some embodiments, the therapeutically effective amount describedherein is provided in one dose. In some embodiments, the therapeuticallyeffective amount described herein is provided in one day.

In other embodiments, a therapeutically effective dosage amount may be,for example, about 0.001 mg/kg weight to 500 mg/kg weight, e.g., fromabout 0.001 mg/kg weight to 400 mg/kg weight, from about 0.001 mg/kgweight to 300 mg/kg weight, from about 0.001 mg/kg weight to 200 mg/kgweight, from about 0.001 mg/kg weight to 100 mg/kg weight, from about0.001 mg/kg weight to 90 mg/kg weight, from about 0.001 mg/kg weight to80 mg/kg weight, from about 0.001 mg/kg weight to 70 mg/kg weight, fromabout 0.001 mg/kg weight to 60 mg/kg weight, from about 0.001 mg/kgweight to 50 mg/kg weight, from about 0.001 mg/kg weight to 40 mg/kgweight, from about 0.001 mg/kg weight to 30 mg/kg weight, from about0.001 mg/kg weight to 25 mg/kg weight, from about 0.001 mg/kg weight to20 mg/kg weight, from about 0.001 mg/kg weight to 15 mg/kg weight, fromabout 0.001 mg/kg weight to 10 mg/kg weight. In some embodiments, thetherapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day.

In still other embodiments, a therapeutically effective dosage amountmay be, for example, about 0.001 mg/kg weight to about 1 mg/kg weight,e.g. from about 0.001 mg/kg weight to about 0.9 mg/kg weight, from about0.001 mg/kg weight to about 0.8 mg/kg weight, from about 0.001 mg/kgweight to about 0.8 mg/kg weight, from about 0.001 mg/kg weight to about0.7 mg/kg weight, from about 0.001 mg/kg weight to about 0.6 mg/kgweight, from about 0.001 mg/kg weight to about 0.5 mg/kg weight, fromabout 0.01 mg/kg weight to about 1 mg/kg weight, from about 0.01 mg/kgweight to about 0.9 mg/kg weight, from about 0.01 mg/kg weight to about0.8 mg/kg weight, from about 0.01 mg/kg weight to about 0.7 mg/kgweight, from about 0.01 mg/kg weight to about 0.6 mg/kg weight, fromabout 0.01 mg/kg weight to about 0.5 mg/kg weight, from about 0.02 mg/kgweight to about 1 mg/kg weight, from about 0.02 mg/kg weight to about0.9 mg/kg weight, from about 0.02 mg/kg weight to about 0.8 mg/kgweight, from about 0.02 mg/kg weight to about 0.7 mg/kg weight, fromabout 0.02 mg/kg weight to about 0.6 mg/kg weight, from about 0.02 mg/kgweight to about 0.5 mg/kg weight, from about 0.03 mg/kg weight to about1 mg/kg weight, from about 0.03 mg/kg weight to about 0.9 mg/kg weight,from about 0.03 mg/kg weight to about 0.8 mg/kg weight, from about 0.03mg/kg weight to about 0.7 mg/kg weight, from about 0.03 mg/kg weight toabout 0.6 mg/kg weight, from about 0.03 mg/kg weight to about 0.5 mg/kgweight, from about 0.04 mg/kg weight to about 1 mg/kg weight, from about0.04 mg/kg weight to about 0.9 mg/kg weight, from about 0.04 mg/kgweight to about 0.8 mg/kg weight, from about 0.04 mg/kg weight to about0.7 mg/kg weight, from about 0.04 mg/kg weight to about 0.6 mg/kgweight, from about 0.04 mg/kg weight to about 0.5 mg/kg weight, fromabout 0.05 mg/kg weight to about 1 mg/kg weight, from about 0.05 mg/kgweight to about 0.9 mg/kg weight, from about 0.05 mg/kg weight to about0.8 mg/kg weight, from about 0.05 mg/kg weight to about 0.7 mg/kgweight, from about 0.05 mg/kg weight to about 0.6 mg/kg weight, fromabout 0.05 mg/kg weight to about 0.5 mg/kg weight. In some embodiments,the therapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day.

In still other embodiments, a therapeutically effective dosage amountmay be, for example, about 0.0001 mg/kg weight to 0.1 mg/kg weight, e.g.from about 0.0001 mg/kg weight to 0.09 mg/kg weight, from about 0.0001mg/kg weight to 0.08 mg/kg weight, from about 0.0001 mg/kg weight to0.07 mg/kg weight, from about 0.0001 mg/kg weight to 0.06 mg/kg weight,from about 0.0001 mg/kg weight to 0.05 mg/kg weight, from about 0.0001mg/kg weight to about 0.04 mg/kg weight, from about 0.0001 mg/kg weightto 0.03 mg/kg weight, from about 0.0001 mg/kg weight to 0.02 mg/kgweight, from about 0.0001 mg/kg weight to 0.019 mg/kg weight, from about0.0001 mg/kg weight to 0.018 mg/kg weight, from about 0.0001 mg/kgweight to 0.017 mg/kg weight, from about 0.0001 mg/kg weight to 0.016mg/kg weight, from about 0.0001 mg/kg weight to 0.015 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.014 mg/kg weight, from about 0.0001 mg/kgweight to 0.013 mg/kg weight, from about 0.0001 mg/kg weight to 0.012mg/kg weight, from about 0.0001 mg/kg weight to 0.011 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.01 mg/kg weight, from about 0.0001 mg/kgweight to 0.009 mg/kg weight, from about 0.0001 mg/kg weight to 0.008mg/kg weight, from about 0.0001 mg/kg weight to 0.007 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.006 mg/kg weight, from about 0.0001 mg/kgweight to 0.005 mg/kg weight, from about 0.0001 mg/kg weight to 0.004mg/kg weight, from about 0.0001 mg/kg weight to 0.003 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.002 mg/kg weight. In some embodiments,the therapeutically effective dose may be 0.0001 mg/kg weight, 0.0002mg/kg weight, 0.0003 mg/kg weight, 0.0004 mg/kg weight, 0.0005 mg/kgweight, 0.0006 mg/kg weight, 0.0007 mg/kg weight, 0.0008 mg/kg weight,0.0009 mg/kg weight, 0.001 mg/kg weight, 0.002 mg/kg weight, 0.003 mg/kgweight, 0.004 mg/kg weight, 0.005 mg/kg weight, 0.006 mg/kg weight,0.007 mg/kg weight, 0.008 mg/kg weight, 0.009 mg/kg weight, 0.01 mg/kgweight, 0.02 mg/kg weight, 0.03 mg/kg weight, 0.04 mg/kg weight, 0.05mg/kg weight, 0.06 mg/kg weight, 0.07 mg/kg weight, 0.08 mg/kg weight,0.09 mg/kg weight, or 0.1 mg/kg weight. The effective dose for aparticular individual can be varied (e.g., increased or decreased) overtime, depending on the needs of the individual.

In some embodiments, the angiotensin (1-7) peptide is administered at aneffective dose ranging from about 1-1,000 μg/kg/day (e.g., ranging fromabout 1-900 μg/kg/day, 1-800 μg/kg/day, 1-700 μg/kg/day, 1-600μg/kg/day, 1-500 μg/kg/day, 1-400 μg/kg/day, 1-300 μg/kg/day, 1-200μg/kg/day, 1-100 μg/kg/day, 1-90 μg/kg/day, 1-80 μg/kg/day, 1-70μg/kg/day, 1-60 μg/kg/day, 1-50 μg/kg/day, 1-40 μg/kg/day, 1-30μg/kg/day, 1-20 μg/kg/day, 1-10 μg/kg/day). In some embodiments, theangiotensin (1-7) peptide is administered at an effective dose rangingfrom about 1-500 μg/kg/day. In some embodiments, the angiotensin (1-7)peptide is administered at an effective dose ranging from about 1-100μg/kg/day. In some embodiments, the angiotensin (1-7) peptide isadministered at an effective dose ranging from about 1-60 μg/kg/day. Insome embodiments, the angiotensin (1-7) peptide is administered at aneffective dose selected from about 1, 2, 4, 6, 8, 10, 15, 20, 25, 30,35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, or 1,000 ug/kg/day.

Dosing Schedules

Various embodiments may include differing dosing regimen. In someembodiments, the angiotensin (1-7) peptide or angiotensin (1-7) receptoragonist is administered via continuous infusion. In some embodiments,the continuous infusion is intravenous. In other embodiments, thecontinuous infusion is subcutaneous. Alternatively or additionally, insome embodiments, the angiotensin (1-7) peptide or angiotensin (1-7)receptor agonist is administered bimonthly, monthly, twice monthly,triweekly, biweekly, weekly, twice weekly, thrice weekly, daily, twicedaily, or on another clinically desirable dosing schedule. The dosingregimen for a single subject need not be at a fixed interval, but can bevaried over time, depending on the needs of the subject.

Combination Therapies

In some embodiments, an Ang (1-7) peptide or angiotensin (1-7) receptoragonist will be used as a part of a combination therapy. It iscontemplated that any known therapeutic or treatment for one or morebrain conditions may be used with one or more Ang (1-7) peptides orangiotensin (1-7) receptor agonists as disclosed herein. Exemplarycompounds that may be used with one or more Ang (1-7) peptides orangiotensin (1-7) receptor agonists as a combination therapy include,but are not limited to, thrombolytic compounds, antioxidants or otherreactive oxygen species agents, interferon beta-1a (e.g. Avonex, Rebif,CinnoVex, ReciGen), interferon beta-1b (Betaseron), glatiramer acetate(Copaxone), mitoxantrone (Novantrone), natalizumab (Tysabri), fingolimod(Gilenya), the first oral drug available, and Teriflunomide (Aubagio),or combinations thereof.

Kits

In some embodiments, the present invention further provides kits orother articles of manufacture which contains an Ang (1-7) peptide, anangiotensin (1-7) receptor agonist or a formulation containing the sameand provides instructions for its reconstitution (if lyophilized) and/oruse. Kits or other articles of manufacture may include a container, asyringe, vial and any other articles, devices or equipment useful inadministration (e.g., subcutaneous, by inhalation). Suitable containersinclude, for example, bottles, vials, syringes (e.g., pre-filledsyringes), ampules, cartridges, reservoirs, or lyo-jects. The containermay be formed from a variety of materials such as glass or plastic. Insome embodiments, a container is a pre-filled syringe. Suitablepre-filled syringes include, but are not limited to, borosilicate glasssyringes with baked silicone coating, borosilicate glass syringes withsprayed silicone, or plastic resin syringes without silicone.

Typically, the container may holds formulations and a label on, orassociated with, the container that may indicate directions forreconstitution and/or use. For example, the label may indicate that theformulation is reconstituted to concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor, for example, subcutaneous administration. In some embodiments, acontainer may contain a single dose of a stable formulation containingan Ang (1-7) peptide or angiotensin (1-7) receptor agonist. In variousembodiments, a single dose of the stable formulation is present in avolume of less than about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml,2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml. Alternatively, a containerholding the formulation may be a multi-use vial, which allows for repeatadministrations (e.g., from 2-6 administrations) of the formulation.Kits or other articles of manufacture may further include a secondcontainer comprising a suitable diluent (e.g., BWFI, saline, bufferedsaline). Upon mixing of the diluent and the formulation, the finalprotein concentration in the reconstituted formulation will generally beat least 1 mg/ml (e.g., at least 5 mg/ml, at least 10 mg/ml, at least 20mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least75 mg/ml, at least 100 mg/ml). Kits or other articles of manufacture mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use. In someembodiments, kits or other articles of manufacture may include aninstruction for self-administration.

EXAMPLES Example 1 Continuous PanCyte Administration

Several animal models have been used to study cerebral ischemia in aneffort to understand its pathophysiology and to identify therapeuticstrategies for minimizing the severity of ischemic damage. Focalischemia brings about localized brain infarction and may be induced bymiddle cerebral artery occlusion (MCAO). A rat model of MCAO has gainedacceptance as a model for hemispheric infarction in humans. After MCAO,a cortical and striatal infarct with temporal and spatial evolutionoccurs within the vascular region supplied by the middle cerebralartery.

In the past several years, ample evidence has been gathered regardingbehavioral assessments in stroke animal studies, including the MCAO ratmodel of ischemic stroke. Behavioral improvement is thought to be areliable parameter for efficacy studies of potential therapeutics.

A desirable treatment for vascular complications of stroke would be anon-invasive means of promoting neovascularization in ischemic tissues.Embodiments of the present invention provide such a therapeutictreatment. Binding of angiotensin (1-7) peptides on the cell surface ofendothelial cells can rescue those cells from apoptosis, induce theirproliferation, migration, and formation of small blood vessels in vitro.

In this example, the MCAO rat model was used to evaluate thedose-dependent efficacy of angiotensin (1-7) peptides, for example,PanCyte, in improving post-occlusion function as measured by severalaccepted behavioral evaluations.

Animal handling was performed according to guidelines of the NationalInstitute of Health (NIH) and the Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC). Animals were housed inpolyethylene cages (5/cage) measuring 35×30×15 cm, with stainless steeltop grill facilitating pelleted food and drinking water in plasticbottle; bedding: steam sterilized clean paddy husk (Harlan, Sani-chipcat#:2018SC+F) was used and bedding material were changed along with thecage at least twice a week. In this example, a total of 60 rats wereused and each rat weighed approximately 300 grams at study initiation.

Animals were fed ad libitum a commercial rodent diet (Teklad CertifiedGlobal 18% Protein Diet cat #: 106S8216). Animals had free access toacidified drinking water (pH between 2.5 and 3.5) obtained from themunicipality supply according to PharmaSeed's SOP No. 214 (WaterSystem). Animals were housed under standard laboratory conditions, airconditioned and filtered (HEPA F6/6) with adequate fresh air supply(Minimum 15 air changes/hour). Animals were kept in a climate controlledenvironment. Animals were kept within a temperatures range ofapproximately 20-24° C. with a relative humidity range of 30-70% and a12 hours light-dark cycle. Animals were inspected on arrival and wereinspected daily for any signs of morbidity or mortality. Animals foundin a moribund condition and animals showing severe pain and enduringsigns of severe distress (such as dyspnea, lateral recumbency,convulsions, plegia or inability to reach food or water) were humanelyeuthanized.

For the purposes of this example, Transient middle cerebral arteryocclusion (tMCAO) procedure Day is defined as “Day 1” in this study. Onthe day of surgery anesthesia were induced with 4% isoflurane in amixture of 70% N₂O and 30% O₂ and maintained with 1.5-2% isoflurane.

The tMCAO procedures were performed according to the method described R.Schmid-Elsaesser et al. Briefly, the right CCA (Common Carotid Artery)was exposed through a midline neck incision and carefully dissected freefrom surrounding nerves and fascia—from its bifurcation to the base ofthe skull. The occipital artery branches of the ECA (External CarotidArtery) were then isolated, and these branches were dissected andcoagulated. The ECA was dissected further distally and coagulated alongwith the terminal lingual and maxillary artery branches, which was thendivided. The ICA (Internal Carotid Artery) was isolated and carefullyseparated from the adjacent vagus nerve, and the pterygopalatine arterywas ligated close to its origin with a 5-0 nylon suture (SMI, Belgium).Next, a 4-0 silk suture was tied loosely around the mobilized ECA stump,and a 4 cm length of 4-0 monofilament nylon suture (the tip of thesuture was blunted by using a flame, and the suture was coated withpolylysine, prior to insertion) was inserted through the proximal ECAinto the ICA and thence into the circle of Willis, effectively occludingthe MCA. The surgical wound was closed and the animals were returned totheir cages to recover from anesthesia. One hour and a half afterocclusion rats were re-anesthetized, the monofilament was withdrawn toallow reperfusion, the surgical wound was closed and rats were returnedto their cages.

Animals were subjected to a modified Modified Neurological Rating Scale(mNRS) at 24 hours post reperfusion. Only animals with an overall scoreof ≧10 are included in this study. Animals were allocated into the testgroups, according to the mNRS results on day 2, in order to have similardistribution of rats performance between groups. Started on day 2, 24hours post-surgery, each animal is implanted subcutaneously with osmoticAlzet pump and is treated by continuous PanCyte administration (2.5mg/ml or 25 mg/ml in PBS; SEQ ID NO:22). See Table 1 for groupallocation:

TABLE 1 Group Allocation Treatment duration Group Treatment Dose (days)Total rats 1 Vehicle 0 49 15 2 PanCyte 50 μg/kg 49 15 (2.5 mg/ml) 3PanCyte 500 μg/kg  49 15  (25 mg/ml) 4 PanCyte 50 μg/kg 14 15 (2.5mg/ml)

Stepping Test (Evaluation at Day 8, Day 15, Day 22, Day 29 and Day 36)

Animals were tested for forelimb akinesia in a stepping test. The animalwas held with its hind limbs fixed with one hand and the forelimb notmonitored with the other, while the unrestrained fore-paw touches atable. The number of adjusting steps were counted while the animal wasmoved sideways along the table surface (85 cm in approximately fiveseconds), in the forehand & backhand direction for both forelimbs. FIG.1 shows the results of the stepping test at days 8, 15, 22, 29 and 36.Four groups were analyzed in this test, a control group (receiving onlyPBS for 49 days), a group receiving 50 μg/kg PanCyte for 49 days, agroup receiving 500 μg/kg PanCyte for 49 days, and a group receiving 50μg/kg PanCyte for 14 days. The data shows that each group receivingadministration of PanCyte enjoyed increased performance of rats by day22 as compared to control animals, and that this effect continuedthrough day 36, with increasing statistical significance. Additionally,the group that was exposed to 50 μg/kg dose of PanCyte for 49 daysperformed significantly better than controls on day 15 as well, whilethe groups exposed to either 500 μg/kg of PanCyte for 49 days or 50μg/kg Pancyte for 14 days, while trending toward improvement, did notreach statistically significant levels at this time point in thisparticular experiment.

Forelimb Placement (Evaluation at Day 8, Day 15, Day 22, Day 29 and Day36)

The limb placing tests were divided into both forelimb and hindlimbtests. For the forelimb-placing test, the examiner held the rat close toa tabletop and scored the rat's ability to place the forelimb on thetabletop in response to whisker, visual, tactile, or proprioceptivestimulation. Similarly, for the hindlimb placing test, the examinerassessed the rat's ability to place the hindlimb on the tabletop inresponse to tactile and proprioceptive stimulation. Separate sub-scoreswere obtained for each mode of sensory input and added to give totalscores (for the forelimb placing test: 0=normal, 12=maximally impaired;for the hindlimb placing test: 0=normal; 6=maximally impaired). Scoreswere given in half-point increments as follows: Forelimb placing test:whisker placing (0-2), visual placing—forward (0-2), —sideways (0-2);tactile placing—dorsal (0-2), —lateral (0-2); proprioceptive placing(0-2); for a total of 0-12. FIG. 2 shows the results of the forelimbplacing test at days 8, 15, 22, 29 and 36. Four groups were analyzed inthis test, a control group (receiving only PBS for 49 days), a groupreceiving 50 μg/kg PanCyte for 49 days, a group receiving 500 μg/kgPanCyte for 49 days, and a group receiving 50 μg/kg PanCyte for 14 days.The data shows that each group receiving administration of PanCyteenjoyed increased performance on this test by day 29 as compared tocontrol animals, and that this effect continued to day 36. Also, thegroup exposed to 50 μg/kg Pancyte for 14 days performed significantlybetter than control animals beginning on day 15, whereas the othertreatment groups, though trending in the same way, did not reachstatistical significance until day 29 in this experiment.

Body Swing Test (Evaluation at Day 8, Day 15, Day 22, Day 29 and Day 36)

The rat was held approximately one inch from the base of its tail. Itwas then elevated to an inch above a surface of a table. The rat washeld in the vertical axis, defined as no more than 10° to either theleft or the right side. A swing was recorded whenever the rat moved itshead out of the vertical axis to either side. Before attempting anotherswing, the rat had to return to the vertical position for the next swingto be counted. Twenty (20) total swings were counted. A normal rattypically has an equal number of swings to either side. Following focalischemia, rats tend to swing to the contralateral side (left side inthis example). Body swing scores are expressed as a percentage ofrightward over total swings. FIG. 3 shows the results of the body swingtest at days 8, 15, 22, 29 and 36. Four groups were analyzed in thistest, a control group (receiving only PBS for 49 days), a groupreceiving 50 μg/kg PanCyte for 49 days, a group receiving 500 μg/kgPanCyte for 49 days, and a group receiving 50 μg/kg PanCyte for 14 days.The data shows that each group receiving administration of PanCyteenjoyed improved performance on the test by day 36 as compared to thecontrol group, with the group receiving 50 μg/kg PanCyte for 14 daysshowed statistical significance beginning on day 29 as compared tocontrol in this experiment. All treatment groups exhibited a trendtoward improved scores beginning on day 8 as compared to the controlgroup.

mNRS Evaluation (Evaluation at Day 1, Day 8, Day 15, Day 22, Day 29 andDay 36)

The Modified Neurological Rating Scale (mNRS) was administered by anindividual who was unaware of the drug/dose given (blind test). The mNRSas administered allows for neuro-scoring on a scale of 0 to 18 possiblepoints. Animals with higher scores showed more severe symptoms anddisability than lower scoring rats. FIG. 4 shows the results of the mNRSevaluation at days 1, 8, 15, 22, 29 and 36. Four groups were analyzed inthis test, a control group (receiving only PBS for 49 days), a groupreceiving 50 μg/kg PanCyte for 49 days, a group receiving 500 μg/kgPanCyte for 49 days, and a group receiving 50 μg/kg PanCyte for 14 days.The data shows that each group receiving administration of PanCyteenjoyed improved performance on the test by day 29 as compared to thecontrol group. In addition to days 29 and 36, the group receiving 50μg/kg PanCyte for 49 days showed statistically improved performance ondays 8 and 15, and the group receiving 500 μg/kg PanCyte for 49 daysshowed statistically improved performance on day 22 in this experiment.

Example 2 Comparison of TXA127, PanCyte, or Linear PanCyteAdministration

The animal model, surgical procedures, and animal care procedures andconditions were as described above for Example 1 unless otherwisespecified. In this example, a total of 105 animals were used, and Table2 shows the group allocation for this study:

TABLE 2 Group Allocation Treatment duration Group Treatment Dose (days)Total rats 1 Vehicle 0 28 15 2 TXA127 500 μg/kg 28 15 3 TXA127 1,000μg/kg 28 15 4 TXA127 500 μg/kg* 28 15 5 PanCyte 500 μg/kg* 28 15 6PanCyte 500 μg/kg 28 15 7 Linear 50 μg/kg* 28 15 PanCyte *= rats treatedby Alzet pump continuous administration subcutaneously

Animals were subjected to a modified Modified Neurological Rating Scale(mNRS) at 24 hours post reperfusion. Only animals with an overall scoreof ≧10 were included in this study. Animals were allocated into the testgroups, according to the mNRS results on day 2, in order to have similardistribution of rats performance between groups. Starting on day 2, 24hours post-surgery, animals in groups 4, 5 and 7 were implantedsubcutaneously with an osmotic Alzet pump and treated with either 500μg/kg TXA127 (SEQ ID NO:1), 500 μg/kg PanCyte (SEQ ID NO:22), or 50μg/kg Linear PanCyte (SEQ ID NO:6). Animals in groups 2, 3 and 6received 500 μg/kg or 1,000 μg/kg TXA127 or 500 μg/kg PanCyte,administered subcutaneously via daily injection. Animals in group 1 weretreated with a daily subcutaneous injection of a PBS (vehicle).

Stepping Test (Evaluation Before Operation, Day 14, Day 21, Day 28, Day35, Day 42 and Day 49)

Animals were tested for forelimb akinesia in a stepping test (ST). Theanimal was held with its hind limbs fixed with one hand and the forelimbnot to be monitored with the other, while the unrestrained fore-pawtouches the table. The number of adjusting steps are counted while theanimal is moved sideways along the table surface (85 cm in approximatelyfive seconds), in the forehand & backhand direction for both forelimbs.FIG. 5 shows treatment with TXA127, PanCyte or Linear Pancytesignificantly improved the performance of treated rats in allexperimental conditions by Day 21 post-surgery, as compared to thevehicle control condition. A trend of improvement is observed as earlyas Day 14 post-surgery. It is of note that although only 50 ug/kg ofLinear Pancyte was administered, the results are substantiallyequivalent to ten times as much TXA127 or PanCyte.

Forelimb Placement (Evaluation Before Operation, Day 14, Day 21, Day 28,Day 35, Day 42 and Day 49)

For the forelimb-placing test, the rat was held close to a tabletop andthe rat's ability to place the forelimb on the tabletop in response towhisker, visual, tactile, or proprioceptive stimulation was scored(0=normal, 12=maximally impaired). Scores were given in half-pointincrements (see below). Typically, there is a slow and steady recoveryof limb placing behavior during the first month after stroke. FIG. 6shows that significant improvement in performance was observed in alltreatment conditions, as compared to vehicle control, beginning on Day14 and continuing through the duration of the study. It again appearsthat the Linear PanCyte group had the best performance, particularlyfrom Day 35 onward, despite being exposed to a far lower dose of agentthan the other experimental groups were.

Body Swing (Evaluation Before Operation, Day 14, Day 21, Day 28, Day 35,Day 42 and Day 49)

Each rat was held approximately one inch from the base of its tail. Itwas then elevated to an inch above a surface of a table. The rat washeld in the vertical axis, defined as no more than 10° to either theleft or the right side. A swing was recorded whenever the rat moves itshead out of the vertical axis to either side. Before attempting anotherswing, the rat must return to the vertical position for the next swingto be counted. Twenty (20) total swings were counted. A normal rattypically has an equal number of swings to either side. Following focalischemia, the rat tends to swing to the contralateral side (left side inthis case). Body swing scores are expressed as a percentage of rightwardover total swings. Often, there is a spontaneous partial recovery ofbody swing scores (toward 50%) during the first month after stroke. FIG.7 shows that the 1,000 μg/kg TXA127, TXA Alzet, PanCyte Alzet, 500 μg/kgPanCyte, and Linear PanCyte groups all showed significant improvement inperformance by Day 28, as compared to the vehicle control. The 500 μg/kgTXA127 group did not show significant results until Day 35. The 1,000μg/kg TXA127, TXA Alzet, PanCyte Alzet, and Linear PanCyte groups allshowed improvement by Day 21, and all experimental groups showed a trendtoward improvement by Day 14. On Day 49, the TXA 1,000 g/kg, TXA Alzet,and Linear PanCyte groups each appeared to perform at a near-normal(uninjured) level.

mNRS Evaluation (Evaluation Before Operation, Day 1, Day 14, Day 21, Day28, Day 35, Day 42 and Day 49)

The Modified Neurological Rating Scale (mNRS) was administered by anindividual who was unaware of the drug/dose given (blind test). The mNRSas administered allows for neuro-scoring on a scale of 0 to 18 possiblepoints. Animals with higher scores showed more severe symptoms anddisability than lower scoring rats. FIG. 8 shows that each experimentalgroup showed significant improvement in performance by Day 14, ascompared to the vehicle control. The observed increased performance wasmaintained for the duration of the study.

These results show that TXA127, PanCyte and Linear PanCyte are allstrong therapeutic candidates with the ability to dramatically improvethe performance of animals post-stroke. In addition to the performancebenefits discussed above, blood flow and blood vessel diameter wasmeasured using Laser Doppler, according to known protocols, in eachnon-Alzet group on Day 50. The results are shown in FIG. 9 and show thatanimals in all assessed treatment groups showed significantly increasedblood vessel diameter and blood flow compared to control animals by Day50. In particular, Linear PanCyte appears to have significantly improvedtherapeutic potential even beyond the other effective treatments testedin this example. These results are particularly surprising since theonly difference between PanCyte and Linear PanCyte appears to be thatPanCyte is cyclized, while Linear PanCyte is not. Cyclization of apeptide is typically thought to allow a peptide to be more effective invivo by making it more resistant to protease degradation. LinearPanCyte, however, is almost equally effective at a lower dose of 50μg/kg (as compared to 500 μg/kg of PanCyte) in this example.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims.

I claim:
 1. A method of treating stroke comprising administering to asubject suffering from stroke an angiotensin (1-7) peptide comprisingthe amino acid sequence Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ IDNO: 1) via systemic administration, wherein the systemic administrationis not intracerebroventricular administration, and wherein theangiotensin (1-7) peptide is administered without the use of modifiedstem cells.
 2. The method of claim 1, wherein the stroke is eitherischemic stroke, hemorrhagic stroke, or a combination thereof.
 3. Themethod of claim 1, wherein the angiotensin (1-7) peptide is administeredvia continuous infusion.
 4. The method of claim 1, wherein theangiotensin (1-7) peptide is administered daily.
 5. The method of claim1, wherein the angiotensin (1-7) peptide is administered twice daily. 6.The method of claim 1, wherein the angiotensin (1-7) peptide isadministered twice per month.
 7. The method of claim 1, wherein theangiotensin (1-7) peptide is administered once per month.
 8. The methodof claim 1, wherein the angiotensin (1-7) peptide is administered at aneffective dose ranging from about 1-1,500 ug/kg/day.
 9. The method ofclaim 1, wherein the angiotensin (1-7) peptide is administered at aneffective dose ranging from about 500-1,500 ug/kg/day.
 10. The method ofclaim 1, wherein the angiotensin (1-7) peptide is administered at aneffective dose ranging from about 800-1,200 ug/kg/day.
 11. The method ofclaim 1, wherein the angiotensin (1-7) peptide comprises one or morechemical modifications to increase protease resistance, serum stabilityand/or bioavailability.
 12. The method of claim 11, wherein the one ormore chemical modifications comprise pegylation, acetylation,glycosylation, biotinylation, or substitution with D-amino acid orun-natural amino acid.
 13. The method of claim 1, wherein the systemicadministration is selected from intravenous administration, subcutaneousadministration, inhalation, intradermal administration, transdermaladministration, transmucosal administration, and/or oral administration.14. The method of claim 13, wherein the systemic administration isintravenous administration.
 15. The method of claim 13, wherein thesystemic administration is subcutaneous administration.
 16. The methodof claim 1, wherein the angiotensin (1-7) peptide is administered as acomponent of a pharmaceutical composition comprising the angiotensin(1-7) peptide and a pharmaceutically acceptable excipient.
 17. Themethod of claim 1, wherein the angiotensin (1-7) peptide is administeredas a part of a combination therapy including at least one additionaltherapeutic or treatment for stroke.
 18. The method of claim 17, whereinthe at least one additional therapeutic or treatment for stroke isselected from a thrombolytic compound, an antioxidant, interferonbeta-1a, interferon beta-1b, glatiramer acetate, mitoxantrone,natalizumab, fingolimod, and Teriflunomide, or combinations thereof.